Dual specific anti-CCL2 antibody

Abstract

The present invention relates to bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their preparation and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases and ophthalmic diseases.

Description

[Technical field]

[0001] The present invention relates to bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their preparation and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases and ophthalmic diseases. [Background technology]

[0002] background The CCL2 / CCR2 axis is the main mediator of the recruitment of immature myeloid cells to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM) building a chemoattractant gradient. Once in the tumor, myeloid-derived suppressor cells (MDSCs) contribute to a pro-tumorigenic environment by secreting / upregulating anti-inflammatory cytokines / receptors that inhibit the initiation of anti-tumor T-cell responses. In this way, MDSCs can reduce or even impair the efficacy of any T-cell activation therapy (Meyer et al., 2014). Thus, specific inhibition of the recruitment of these immature myeloid cells enhances the efficacy of checkpoint inhibitors, T-cell bispecific antibodies (TCBs) or other cancer immunotherapies (CITs). Moreover, CCL2 is also involved in promoting angiogenesis, metastasis and tumor growth, suggesting that neutralization of CCL2 may contribute to some anti-tumor interventions.

[0003] Targeting CCL2, as opposed to its receptor, specifically inhibits undesirable CCL2-mediated effects, sparing the same receptor (CCR2) that is involved in the recruitment of other immune cell populations such as Th1 and NK cells, but which may signal through different ligands (e.g., CCL7, CCL8, CCL13).

[0004] Clinically, CCL2 has been the preferred antibody target in several studies aimed at neutralizing its elevated levels caused by different inflammatory diseases such as rheumatoid arthritis (Haringman et al, Arthritis Rheum. 2006 Aug;54(8):2387-92), idiopathic pulmonary fibrosis (Raghu et al, Eur Respir J. 2015 Dec;46(6):1740-50), diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32:307-315), and cancer (Sandhu et al, Cancer Chemother Pharmacol. 2013 Apr;71(4):1041-50). However, its high synthesis rate, together with the observed high in vivo antibody-antigen dissociation constants (KD), have proven to be a major obstacle preventing the inhibition of free CCL2 by conventional antibodies at clinically viable doses (Fetterly et al, J Clin Pharmacol. 2013 Oct;53(10):1020-7).

[0005] CCL2 neutralization appears to be more clearly relevant in patients with elevated serum levels of CCL2, which has been observed in several cancer types, such as breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer. However, even patients within these indications who do not present this serology, but whose tumors are highly infiltrated with immune cells of myeloid lineage, can greatly benefit from this novel therapy due to the many roles that CCL2 plays in the tumor context, as discussed above.

[0006] Igawa et al, Immunological Reviews 270 (2016) 132-151, describe a Sweeping technology whereby the generated antibodies have pH-dependent CDRs (resulting in antigen degradation due to antibody-antigen dissociation in acidic endosomes) and an engineered Fc portion with optimized isoelectric point (pI) and enhanced binding to Fc gamma RIIb (facilitating cellular uptake of immune complexes) and moderate affinity for neonatal Fc receptors to maintain an acceptable pharmacokinetic profile. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to certain bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their preparation and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases and ophthalmic diseases. [Means for solving the problem]

[0008] The present invention provides a bispecific antibody comprising a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site which (specifically) binds to a second, different epitope on human CCL2, The bispecific antibody a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Including, A) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or B) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or C) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or D) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or E) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or F) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or G) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or H) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or I) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or J) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or K) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or L) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or M) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or N) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or O) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or P) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; A bispecific antibody is provided, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.

[0009] One embodiment of the present invention is the bispecific antibody described above, i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; A bispecific antibody, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.

[0010] One embodiment of the present invention is the bispecific antibody described above, i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; A bispecific antibody, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO:94.

[0011] In one embodiment of the invention, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG isotype, preferably the human IgG1 isotype.

[0012] In one embodiment of the invention, the bispecific antibody comprises: i) Blocking the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC 50 =0.5 nM), and / or ii) inhibited CCL2-mediated chemotaxis of myeloid cells in vitro (IC 50 = 1.5 nM), and / or iii) cross-reactive with cynomolgus monkey (cyno) and human CCL2;

[0013] In one embodiment of the invention, the bispecific antibody comprises: The bispecific antibody is not cross-reactive to other CCL homologues (showing 100-fold less binding to other CCL homologues (selected from the group of CCL8, CCL7 and CCL13) compared to binding to CCL2).

[0014] In one embodiment of the invention, the bispecific antibody comprises: The bispecific antibody binds to a first and a second epitope on human CCL2 in an ion-dependent manner.

[0015] In one embodiment of the invention, the bispecific antibody comprises: The bispecific antibody binds to human CCL2 in a pH-dependent manner, with both the first antigen-binding site and the second antigen-binding site binding to CCL2 with higher affinity at neutral pH than at acidic pH.

[0016] In one embodiment of the invention, the bispecific antibody comprises: The bispecific antibody binds to human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

[0017] In one embodiment of the invention, the constant heavy domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L235W, G236N, H268D, Q295L, K326T and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) N434A (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0018] In one embodiment of the invention, the constant heavy domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0019] The present invention further provides isolated nucleic acid encoding a bispecific antibody according to the invention described herein.

[0020] The present invention further provides a host cell containing such nucleic acid.

[0021] The invention further provides a method for producing a bispecific antibody comprising culturing such a host cell to produce the bispecific antibody.

[0022] The present invention further provides a pharmaceutical formulation comprising a bispecific antibody according to the invention as described herein and a pharma- ceutically acceptable carrier.

[0023] The present invention further provides a bispecific antibody according to the invention as described herein for use as a medicament.

[0024] The present invention further provides a bispecific antibody according to the invention as described herein for use in the treatment of cancer.

[0025] The present invention further provides a bispecific antibody according to the invention as described herein for use in the treatment of an inflammatory or autoimmune disease.

[0026] The present invention further provides the use of a bispecific antibody according to the invention described herein in the manufacture of a medicament.

[0027] In one aspect, the invention is based in part on the discovery that the bispecific antibodies described herein use different anti-CCL2 antigen binding sites as the first and second antigen binding sites / moieties. These bispecific anti-CCL2 antibodies have the ability to bind with high specificity to a particular epitope of CCL2 and specifically inhibit the binding of CCL2 to its receptor CCR2. They show improved immune complex formation and improved CCL2 inhibition in vivo compared to monospecific antibodies. The specific bispecific anti-CCL2 antibodies in the contour body format described herein further exhibit advantageous properties such as low viscosity, which allows for highly concentrated solutions suitable for e.g. subcutaneous administration. [Brief description of the drawings]

[0028] [Figure 1] Surface plasmon resonance (Biacore®) sensorgrams showing binding of monospecific anti-CCL2 antibodies (CNTO888 (=CNTO), 1A5, 1G9 and humanized 11K2 (=11k2) to recombinant CCL2 and CCL2 homologues. [Figure 2a] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody CNTO888-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody CNTO888-SG105 (Fc receptor binding silencing IgG1). [Figure 2b] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 11K2-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silencing IgG1). [Figure 2c] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody ABN912-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody ABN912-SG105 (Fc receptor binding silencing IgG1). [Figure 2d]a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1A4-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1A4-SG105 (Fc receptor binding silencing IgG1). [Figure 2e] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1A5-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1A5-SG105 (Fc receptor binding silencing IgG1). [Figure 2f] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1G9-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 1G9-SG105 (Fc receptor binding silencing IgG1). [Figure 2g] a) Solid line: serum concentration of hCCL2 over time following iv injection into FcRn transgenic mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 2F6-SG1 (wild type IgG1), or b) dotted line: 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg monospecific anti-CCL2 antibody 2F6-SG105 (Fc receptor binding silencing IgG1). [Figure 3a]a) Solid line: Time course of serum total mouse CCL2 concentration (Figure 3a) and antibody-time profile (Figure 3b) following iv injection in mice of 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG1 (wild type IgG1) and b) dotted line: 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silencing IgG1). [Figure 3b] a) Solid line: Time course of serum total mouse CCL2 concentration (Figure 3a) and antibody-time profile (Figure 3b) following iv injection in mice of 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG1 (wild type IgG1) and b) dotted line: 20 mg / kg of monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silencing IgG1). [Figure 4a] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 11K2 / / 1G9-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 11K2 / / 1G9-PGLALA (Fc receptor binding silencing IgG1). [Figure 4b] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody CNTO888 / / 11K2-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody CNTO888 / / 11K2-PGLALA (Fc receptor binding silencing IgG1). [Figure 4c]a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody CNTO888 / / 1G9-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 11K2 / / 1G9-PGLALA (Fc receptor binding silencing IgG1). [Figure 4d] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody CNTO888 / / 1A5-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody CNTO888 / / 1A5-PGLALA (Fc receptor binding silencing IgG1). [Figure 4e] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A5 / / 1G9-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A5 / / 1G9-PGLALA (Fc receptor binding silencing IgG1). [Figure 4f] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 11K2 / / 2F6-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 11K2 / / 2F6-PGLALA (Fc receptor binding silencing IgG1). [Figure 4g] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody ABN912 / / 11K2-WT IgG1 (wild-type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody ABN912 / / 11K2-PGLALA (Fc receptor binding silencing IgG1). [Figure 4h] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A4 / / 2F6-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A4 / / 2F6-PGLALA (Fc receptor binding silencing IgG1). [Figure 4i] a) Solid line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A5 / / 2F6-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line: serum concentration of hCCL2 over time following iv injection into Balb / c mice of preformed immune complexes consisting of 0.1 mg / kg human CCL2 (hCCL2) and 20 mg / kg bispecific anti-CCL2 antibody 1A5 / / 2F6-PGLALA (Fc receptor binding silencing IgG1). [Figure 5a] Biacore® sensorgrams showing the binding profiles of the four modified 11K2 and four CNTO888 variants to monomeric CCL2 at pH 7.4 (black lines) and pH 5.8 (grey lines) and to the 16 bispecific anti-CCL2 antibodies CKLO01 to CKLO16 resulting from the combined antigen-binding portions of the four modified 11K2 and four CNTO888 variants, respectively. [Figure 5b]Biacore® sensorgrams showing the binding profiles of the four modified 11K2 and four CNTO888 variants to monomeric CCL2 and the 16 bispecific anti-CCL2 antibodies CKLO01 to CKLO16 resulting from the combined antigen-binding portions of the four modified 11K2 and four CNTO888 variants, respectively. An additional dissociation phase at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. [Figure 6] Biacore® sensorgrams showing the binding profiles of bispecific anti-CCL2 antibodies CKLO01, CKLO02, CKLO03 and CKLO04 to monomeric CCL8 at pH 7.4 (black line) and pH 5.8 (grey line). [Figure 7a] Serum concentrations of hCCL2 over time after injection of preformed immune complexes consisting of hCCL2 and bispecific anti-CCL2 antibodies (parent CNTO / / 11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03 and CKLO04) into SCID mice. [Figure 7b] Serum concentrations of hCCL2 over time after injection of preformed immune complexes consisting of hCCL2 and CKLO03 (containing IgG1 wild-type Fc) or CKLO03-SG1099 (CKLO03 with enhanced pI Fc) into SCID mice. [Figure 8]Chemotaxis assay: Bispecific anti-CCL2 antibodies with identical CDRs and variable regions VH / VL, i.e. CKLO2-IgG1 wild type and CKLO2-SG1095, but with different Fc parts, are able to inhibit migration of THP-1 cells with identical potency (IC50=0.2 μg / ml; FIG. 8, left panel). Similarly, CCL2-0048, the parent unmodified bispecific antibody CNTO888 / 11k2k2 IgG1 of CKLO2, which is pH independent, also shows an IC50 of 0, 2 μg / ml, a phenomenon that does not occur in this assay, since pH dependence is important for antigen scavenging. The corresponding monospecific antibodies CNTO888 IgG1 and humanized 11k2 IgG1 show IC50 values ​​of 0.3 and 0.7 μg / ml, respectively, whereas the huIgG1 isotype control shows no inhibition (FIG. 8, right panel). [Figure 9] In vivo antitumor activity in genetically engineered mouse models. Treatment of mouse tumor models with Mabs CKLO2-IgG1 (Fc wild-type IgG1) and CKLO2-SG1099 (=CKLO2 pI enhanced). Tumor volume (left), tumor weight (middle) and M-MDSC infiltrate (right) at the end of the study. (Vehicle in black, CKLO2 wild-type IgG1 in grey and CKLO2 pI enhanced Fc (CKLO2-SG1099) in white bars / dotted line) [Figure 10] Serum total (left) and free (right) CCL2 levels during in vivo antitumor activity studies (see efficacy in Figure 9) treatment with bispecific anti-CCL2 antibodies (vehicle in black, CKLO2 wild type IgG1 in grey, and pI enhanced Fc (CKLO2-SG1099) in white bars / dotted line). [Figure 11]Proof-of-concept study of CCL2 sweep efficiency in cynomolgus monkeys. Total antibody concentration-time profiles in serum of cynomolgus monkeys; Left panel: Mean concentration-time profiles of four antibodies over 7 days; Group 1: Monospecific CNTO888-SG1 (= IgG1 wild type) anti-CCL2 antibody as control for maximum total CCL2 accumulation (n = 3 animals); Group 2: Biparatopic anti-CCL2 antibody CKLO2-SG1 (IgG1 wild type) with pH-dependent target binding but no Fc modification (n = 3); Group 3: Biparatopic anti-CCL2 antibody CKLO2-SG1100 with pH-dependent target binding and Fc-pI as well as further modifications (n ​​= 4), and Group 4: Biparatopic anti-CCL2 antibody CKLO2-SG1095 with pH-dependent target binding, Fc-pI and FcγRIIb affinity enhancement and further modifications (n ​​= 4); Right panel: Individual concentration-time profile of individual 4 (group 2) over the PK study period (70 days) is shown. [Figure 12] Proof-of-concept study of CCL2 sweep efficiency in cynomolgus monkeys. Total CCL2 concentration-time profiles in serum of cynomolgus monkeys; Left panel: Mean total CCL2 concentration-time profiles of four antibodies over 7 days; Right panel: Individual total CCL2 concentration-time profile of individual 4 (group 2) over the PK study period (70 days). [Figure 13] Serum free CCL2 concentration-time profiles in cynomolgus monkeys; left panel: average free CCL2 concentration-time profiles of the four antibodies over a 7-day period; right panel: individual free CCL2 concentration-time profiles of individual 4 (group 2) over the PK study period (70 days); for samples below the limit of detection, a value of 0.01 ng / mL (lower limit of quantification) was used to calculate the average profiles. [Figure 14]PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Total CKL02-SG1095 concentration-time profile in serum of cynomolgus monkeys (CKL02-SG1095 treatment at different concentrations (groups 1-3)); Left panel: shows the mean concentration-time profile (n=4) for three dose levels over 7 days; Right panel: shows individual concentration-time profile of two ADA-negative individual animals (25 mg / kg dose group) over the study period (98 days). [Figure 15] PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Total CCL2 concentration-time profiles in serum of cynomolgus monkeys under CKL02-SG1095 treatment at different concentrations (groups 1-3) and comparison with CNTO888-SG1 treatment (group 4); Left panel: presents the mean total CCL2 concentration-time profiles (error bars indicate SD) of the four test groups over 7 days; Right panel: shows individual total CCL2 concentration-time profiles of ADA-negative animals in group 3 (n=2, error bars indicate range) and group 4 (n=3, error bars indicate SD) over the PK study period (98 days). [Figure 16] PK / PD study of CCL2 sweep efficiency in cynomolgus monkeys. Serum free CCL2 concentration-time profiles of cynomolgus monkeys; Left panel: Mean free CCL2 concentration-time profiles (error bars indicate SD) of the four study groups over 7 days (compared to CKL02-SG1095 treatment (groups 1-3) and CNTO888-SG1 treatment (group 4) at different concentrations); Right panel: Mean free CCL2 concentration-time profiles of ADA-negative animals in group 3 (n=2, error bars indicate range) and group 4 (n=3, error bars indicate SD) over the PK study period (70 days); For samples below the detection limit, a value of 0.01 ng / mL (lower limit of quantification) was used to calculate the mean profile. [Figure 17] FIG. 1: Exemplary scheme of bispecific anti-CCL2 antibodies of the invention (so-called contour body (CB) format). [Figure 18] SEC complex: SEC of CCL2 complex with biparatopic antibodies P1AF8139 (abbreviated as 39) and P1AF8143 (abbreviated as 43) [Figure 19a] Figures 19A-C: Cellular uptake of CCL2 via Fc gamma-IIa-FcRIIa. Figure 19A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 19B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 19C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (=CKLO2-SG1 (=IgG1 wt), and absence of antibody as a reference set. [Figure 19b] Figures 19A-C: Cellular uptake of CCL2 via Fc gamma-IIa-FcRIIa. Figure 19A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 19B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 19C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (=CKLO2-SG1 (=IgG1 wt), and absence of antibody as a reference set. [Figure 19c] Figures 19A-C: Cellular uptake of CCL2 via Fc gamma-IIa-FcRIIa. Figure 19A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 19B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 19C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (=CKLO2-SG1 (=IgG1 wt), and absence of antibody as a reference set. [Figure 20a] Figures 20A-C: Cellular uptake of CCL2 via Fc gamma-IIb-FcRIIb. Figure 20A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 20B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 20C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (= CKLO2-SG1 (= IgG1 wt), and absence of antibody as a reference set. [Figure 20b]Figures 20A-C: Cellular uptake of CCL2 via Fc gamma-IIb-FcRIIb. Figure 20A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 20B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 20C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (= CKLO2-SG1 (= IgG1 wt), and absence of antibody as a reference set. [Figure 20c] Figures 20A-C: Cellular uptake of CCL2 via Fc gamma-IIb-FcRIIb. Figure 20A: Y-shaped biparatopic antibodies with reference antibody P1AD8325, Figure 20B: Contose bodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325, and Figure 20C: Antibodies P1AD8325, CNTO888-IgG1, CKLO2 (= CKLO2-SG1 (= IgG1 wt), and absence of antibody as a reference set. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The present invention relates to bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2, pharmaceutical compositions thereof, their preparation and use as medicaments for the treatment of cancer, inflammatory diseases, autoimmune diseases and ophthalmic diseases.

[0030] In one embodiment of the invention, the bispecific anti-CCL2 antibody comprises a first antigen-binding site which (specifically) binds to a first epitope on human CC2 and a second, different antigen-binding site which (specifically) binds to a second, different epitope, the bispecific anti-CCL2 antibody comprising: a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Includes.

[0031] As used herein, the term "CCL2", also referred to as "MCP-1", "human CCL2" refers to the 76 amino acid sequence referenced in NCBI record accession number NP_002973 and variously known as CCL2, MCP-1 (monocyte chemotactic protein 1), SMC-CF (smooth muscle cell chemotactic factor), LDCF (lymphocyte-derived chemotactic factor), GDCF (glioma-derived monocyte chemotactic factor), TDCF (tumor-derived chemotactic factor), HCl1 (human cytokine 11), MCAF (monocyte chemotactic and activating factor). The gene symbol is SCYA2, the JE gene on human chromosome 17, with the new name CCL2 (Zlotnik, Yoshie 2000. Immunity 12:121-127). JE is the mouse homolog of human MCP-1 / CCL2.

[0032] Handel and others (Biochemistry. 1996;35:6569-6584) determined the solution structure of the CCL2 dimer. These studies showed that the secondary structure of CCL2 consists of four β-sheets. Furthermore, the residues responsible for the dimerization interface of CCL2 have been described by Zhang and Rollins (Mol Cell Biol. 1995;15:4851-4855). The protein complex appears elongated with the two monomers oriented to form a large pocket. The structures of two crystal forms, the so-called I and P forms, of monomeric and dimeric CCL2 have also been determined (Lubkowski et al., Nat Struct Biol. 1997;4:64-69). Paolini et al, (J Immunol. 1994 Sep 15;153(6):2704-17) described that MCP1 / CCL2 exist as monomers in physiologically relevant concentrations: by analyzing rec.CCL2 protein (purchased from Peprotech) by size-exclusion HPLC, sedimentation equilibrium ultracentrifugation and chemical cross-linking, they were able to show that the weight fraction of MCP-1 monomers and dimers depends on their cooperation in vitro. Finally, Seo and co-workers (J Am Chem Soc. 2013 Mar 20;135(11):4325-32) were able to show by ion mobility mass spectrometry the presence of injected CCL2 in both monomeric and dimeric forms under physiological conditions.

[0033] Thus, "wild type CCL-2" (wt CCL2) can exist as a monomer, but can also in fact form dimers at physiological concentrations. This monomer-dimer equilibrium is certainly different and needs to be carefully considered for all described in vitro experiments in which different concentrations may be used. To avoid uncertainty, we created a point mutation CCL2 variant: the "P8A" variant of CCL2 carries a mutation in the dimerization interface and is therefore unable to form dimers resulting in a defined pure CCL2 monomer. In contrast, the "T10C" variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20;135(11):4325-32).

[0034] The CCL2 / CCR2 axis is the main mediator of the recruitment of immature myeloid cells to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM) building a chemoattractant gradient. Once in the tumor, myeloid-derived suppressor cells (MDSCs) contribute to a pro-tumorigenic environment by secreting / upregulating anti-inflammatory cytokines / receptors that inhibit the initiation of anti-tumor T-cell responses. In this way, MDSCs may reduce or even impair the efficacy of any T-cell activation therapy (Meyer et al., 2014). Thus, specific inhibition of the recruitment of these immature myeloid cells enhances the efficacy of checkpoint inhibitors, T-cell bispecifics and cancer immunotherapy. Moreover, CCL2 is also involved in promoting angiogenesis, metastasis and tumor growth, suggesting that neutralization of CCL2 may contribute to some anti-tumor interventions.

[0035] Targeting CCL2, as opposed to its receptor, specifically inhibits undesirable CCL2-mediated effects, sparing the same receptor (CCR2) that is involved in the recruitment of other immune cell populations such as Th1 and NK cells, but which may signal through different ligands (e.g., CCL7, CCL8, CCL13).

[0036] Clinically, CCL2 has been the preferred antibody target in several studies aimed at neutralizing its elevated levels caused by different inflammatory diseases such as rheumatoid arthritis (Haringman et al., 2006), idiopathic pulmonary fibrosis (Raghu et al., 2015), diabetic nephropathy (Menne et al., 2016), and cancer (Sandhu et al., 2013). However, its high synthesis rate, together with the observed high in vivo antibody-antigen dissociation constant (KD), has proven to be the main obstacle preventing the inhibition of free CCL2 by conventional antibodies at clinically viable doses (Fetterly et al., 2013).

[0037] CCL2 neutralization appears to be more clearly relevant in patients with elevated serum levels of CCL2, which has been observed in several cancer types, such as breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer. However, even patients within these indications who do not present this serology, but whose tumors are highly infiltrated with immune cells of myeloid lineage, can greatly benefit from this novel therapy due to the many roles that CCL2 plays in the tumor context, as discussed above.

[0038] As used herein, an antibody that "binds to human CCL2," "specifically binds to human CCL2," "binds to human CCL2," or "anti-CCL2" is an antibody that is more than 5.0×10 -8 K in mol / l D value, in one embodiment 1.0x10 -9 K in mol / l D Value, in one embodiment 5.0x10 -8 mol / l~1.0x10 -13 K in mol / l D The term refers to an antibody that specifically binds to the human CCL2 antigen with a binding affinity of 0.1 μg / mL.

[0039] Binding affinity is determined in standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), for example using a construct comprising the CCL2 extracellular domain (e.g., in its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined in standard binding assays using an exemplary soluble CCL2.

[0040] The specificity of an antibody refers to the selective recognition of the antibody for a particular epitope of an antigen. For example, natural antibodies are monospecific.

[0041] As used herein, the term "monospecific" antibody denotes an antibody that has one or more binding sites each of which binds to the same epitope of the same antigen.

[0042] As used herein, the terms "bispecific antibody that binds to (human) CCL2", "biparatopic antibody that binds to (human) CCL2", "bispecific anti-CCL2 antibody", "biparatopic anti-CCL2 antibody" mean that the antibody can specifically bind to at least two different epitopes on (human) CCL2. Typically, such a bispecific antibody comprises two different antigen binding sites (two different paratopes), each specific for a different epitope of (human) CCL2. In certain embodiments, the bispecific antibody can bind to two different non-overlapping epitopes on CCL2, which means that the two different antigen binding sites do not compete for binding to CCL2.

[0043] As used herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0044] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0045] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0046] As used herein, the term "valency" refers to the presence of a specified number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and no more than one) antigen-binding site in an antibody that is specific for the antigen.

[0047] The term "antigen-binding site" refers to a site or region of an antibody, i.e., one or several amino acid residues, that provide interaction with an antigen. For example, an antigen-binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). In one embodiment, an antigen-binding site of an antibody comprises amino acid residues from VH and VL. A native immunoglobulin molecule typically comprises two antigen-binding sites; a Fab molecule typically has one antigen-binding site. An "antigen-binding portion" refers to a polypeptide molecule that comprises an antigen-binding site that specifically binds to an antigenic determinant. Antigen-binding portions include antibodies and fragments thereof as further defined herein. Particular antigen-binding portions comprise the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding portion may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include any of the two isotypes: kappa and lambda.

[0048] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety / site binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0049] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0050] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Preferably, the bispecific antibodies of the invention are of the human IgG isotype, more preferably of the human IgG1 isotype. The terms IgG isotype and IgG1 isotype as used herein refer to human IgG isotype and human IgG1 isotype. Typically, different IgG isotypes exist in slightly different allotypic forms based on allelic variation between IgG subclasses (see Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17). An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0051] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by a host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may contain a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein, which is comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein, which is comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions of the invention, such as pharmaceutical compositions described herein, comprise a collection of antibodies or bispecific antibodies of the invention. The population of antibodies or bispecific antibodies may include molecules with full-length heavy chains and molecules with truncated variant heavy chains.The population of antibodies or bispecific antibodies may consist of a mixture of molecules with full-length heavy chains and molecules with truncated variant heavy chains, where at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have truncated variant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain as specified herein, which comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain as specified herein, which comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such compositions comprise antibodies or bispecific antibody populations comprised of molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein, molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat), molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In a particular embodiment, the lysine at position 447, numbered according to EU index of Kabat, is replaced by a glycine (K447G) mutation and the molecule comprises an additional C-terminal glycine-glycine dipeptide (G446 and G447, numbering according to EU index of Kabat).Unless otherwise specified herein, the numbering of amino acid residues in the Fc region and constant region is "according to the EU numbering system" as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above), also referred to as "numbering according to Kabat's EU index" or "Kabat EU numbering". As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0052] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally appear in a VH (or VL) in the following sequence: FR-H1(L1)-CDR-H1(L1)-FR-H2(L2)-CDR-H2(L2)-FR-H3(L3)-CDR-H3(L3)-FR-H4(L4).

[0053] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc region as defined herein.

[0054] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or of an antibody derived from a non-human source that utilizes the human antibody repertoire, or to sequences encoding other human antibodies. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0055] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (see above). In one embodiment, for VH, the subgroup is subgroup III in Kabat et al. (see above).

[0056] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to the CDRs of a non-human antibody and all or substantially all of the FRs corresponding to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0057] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58 (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b) and / or (c) comprising CDR amino acid residues 24 to 34 (CDR-L1), 50 to 56 (CDR-L2), 89 to 97 (vL3), 31 to 35 (CDR-H1), 50 to 63 (CDR-H2) and 95 to 102 (CDR-H3).

[0058] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0059] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans, non-human primates such as monkeys), rabbits, rodents (e.g., mice, rats, etc.), etc. In certain embodiments, the individual or subject is a human.

[0060] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0061] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0062] "Isolated nucleic acid encoding a mono- or bispecific anti-CCL2 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations within a host cell.

[0063] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0064] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0065] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which may contain information regarding the indications, use, dosage, administration, concomitant therapy, contraindications and / or warnings pertaining to such therapeutic product.

[0066] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0067] In situations where ALIGN-2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, then the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0068] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to a subject to which the formulation will be administered.

[0069] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0070] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease symptoms, and improving recovery or prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to delay the progression of disease.

[0071] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (CDR). (See, for example, Kindt, TJ et al. Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds the antigen. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0072] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as autonomously replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0073] I. Compositions and Methods In one aspect, the invention is based in part on the discovery that the bispecific antibodies described herein use different anti-CCL2 antigen binding sites as the first and second antigen binding sites / moieties. These bispecific anti-CCL2 antibodies have the ability to bind with high specificity to a particular epitope of CCL2 and specifically inhibit the binding of CCL2 to its receptor CCR2. They show improved immune complex formation and improved CCL2 inhibition in vivo compared to monospecific antibodies. The specific bispecific anti-CCL2 antibodies in the contour body format described herein further exhibit advantageous properties such as low viscosity, which allows for highly concentrated solutions suitable for e.g. subcutaneous administration.

[0074] Bispecific anti-CCL2 antibody bispecific antibody Bispecific antibodies as described herein are monoclonal antibodies that have different binding specificities for at least two different epitopes on CCL2.

[0075] Techniques for making multispecific and bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), using general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and by preparation of trispecific antibodies, for example as described in Tutt et al. J. Immunol. 147:60 (1991).

[0076] Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain antigen-binding sites that bind to CCL2 and to another different antigen or two different epitopes of CCL2 (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0077] Multispecific antibodies can also be provided in asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e. by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253) or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20) (also called CrossMab). Asymmetric binding arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to induce correct Fab pairing. See, for example, WO 2016 / 172485.

[0078] A variety of additional molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0079] Bispecific antibody formats of the invention: Preferred bispecific antibodies of the invention are of the following format:

[0080] Bispecific antibodies are a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, the first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Includes.

[0081] This basic antibody Fc domain, including the "contorsbody" (CB) format, is described, for example, in Guy J. Georges et al, Computational and Structural Biotechnology Journal Volume 18, 2020, Pages 1210-1220. See also the scheme in Figure 17, which shows an example of a bispecific contour body format with different regions and components. The black circle between the CH3 domains is any heterodimerization that facilitates modification / mutation of the CH3 domain (e.g., knob to hole, described in more detail below in the section referring to heterodimerization that facilitates Fc modifications).

[0082] The term "polypeptide linker" refers to a linker of natural and / or synthetic origin. A polypeptide linker consists of a linear chain of amino acids, the 20 natural amino acids being monomeric building blocks linked by peptide bonds. The chain has a length of 1 to 15 amino acid residues. A polypeptide linker may comprise a repeating amino acid sequence or sequences of naturally occurring polypeptides. A polypeptide linker has the function of ensuring that the antibody domains of the bispecific contour body are able to fold correctly and be properly presented, thereby enabling them to carry out their biological activity. Preferably, the polypeptide linker is a "synthetic peptide linker", which is indicated to be rich in glycine and / or serine residues. These residues are arranged in small repeating units, for example of up to 5 amino acids.

[0083] The linkers L1 and L2 are preferably glycine-serine linkers. The serine residue provides some polarity to the chain to provide solubility to the linker. As described in WO2019233842, the transition between the linker segment and the fusion protein fragment is preferably free of a GS motif due to the possibility of post-translational modification, i.e. O-glycosylation. The contour body described in this application is then composed of a terminal glycine. Variations in length / composition were tested. Any combination of L1 and L2 is contemplated. The repeated glycines are limited to a maximum of four consecutive glycines. If the C-terminal segment of a domain fused to another via a linker is made of, for example, one or two glycines (e.g., if the C-terminus of the CH3 domain is modified to terminate in a glycine or terminate in two glycines), these one or two glycines must take into account the limit of a maximum of four consecutive glycines. Up to six further naturally occurring amino acids may be added at the amino and / or carboxy termini of the multimeric units. Exemplary linkers having a length of 10 amino acids are for example selected from the group GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185), GGSGGSGGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), GGGGSGGSGG (SEQ ID NO: 191), preferably GGSGGGGSGG (SEQ ID NO: 188). Similarly, further linkers having a length of 5 to 9 amino acids or a length of 11 to 15 amino acids can be constructed.

[0084] Fc Domains and Modifications In certain embodiments, a bispecific antibody of the invention comprises an Fc domain composed of a first and a second subunit, it being understood that the characteristics of the Fc domain described herein in relation to a bispecific antibody may equally be applied to the Fc domain comprised in an antibody of the invention.

[0085] The Fc domain of a bispecific antibody consists of a pair of polypeptide chains that contain the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, a bispecific antibody of the invention does not contain more than one Fc domain.

[0086] In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU numbering), in particular the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange in vivo of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgG1 Fc domain.

[0087] The Fc domains of IgG isotypes are characterized by various properties based on, for example, their interactions with Fc gamma receptors or neonatal Fc receptors (FcRn) (see, for example, Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17).

[0088] Fc domain modifications that promote heterodimerization The bispecific antibodies according to the invention comprise different antigen-binding moieties that can be fused to one or the other of the two subunits of the Fc domain, which are thus typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antibodies in recombinant production, it is advantageous to introduce modifications in the Fc domain of the bispecific antibody that promote the association of the desired polypeptides.

[0089] Thus, in a particular embodiment, the Fc domain of the bispecific antibody according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The longest site of protein-protein interaction between the two subunits of a human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0090] There are several approaches to modifications in the CH3 domain of the Fc domain to enhance heterodimerization, which are fully described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) is directed not to homodimerize with itself but to heterodimerize with another CH3 domain that has been engineered in a complementary manner (so that the first CH3 domain and the second CH3 domain heterodimerize and no homodimers are formed between the two first CH3 domains or the two second CH3 domains). These different approaches for improved heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications in bispecific antibodies that reduce heavy / light chain mispairing and Bence-Jones type by-products (e.g., swapping / replacing VH and VL in one binding arm and introducing substitutions of charged amino acids with opposite charges at the CH1 / CL interface).

[0091] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0092] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the cavity to promote heterodimer formation and discourage homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the protrusion are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0093] Thus, in certain embodiments, in the CH3 domain of a first subunit of the Fc domain of a bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit which can be repositioned in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be repositioned.

[0094] Preferably, the amino acid residue having a larger side chain mass is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0095] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0096] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis or by peptide synthesis.

[0097] In a specific embodiment, in the (CH3 domain of) the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the (CH3 domain of) the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0098] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the replacement of the serine residue at position 354 with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine ​​residues allows the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0099] In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0100] In certain embodiments, an antigen binding moiety that binds a second antigen is fused (optionally via a first antigen binding moiety that binds CCL2 and / or a peptide linker) to a first subunit of an Fc domain (including a "knob" modification). Without wishing to be bound by theory, the fusion of an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) to a knob-containing subunit of an Fc domain (further) minimizes the generation of antibodies comprising two antigen binding moieties that bind to an activating T cell antigen (steric clash of the two knob-containing polypeptides).

[0101] Other techniques for CH3 modifications to enhance heterodimerization are contemplated as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1 870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0102] In one embodiment, the heterodimerization approach described in EP 1870459 is used instead. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the bispecific antibody of the invention is the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain) and D399K, E357K in the other CH3 domain of the Fc domain (numbering according to Kabat EU index).

[0103] In another embodiment, a bispecific antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0104] In another embodiment, a bispecific antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said bispecific antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to Kabat EU index).

[0105] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to Kabat EU index). In a further embodiment, the first CH3 domain comprises the further amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises a further amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbering according to Kabat EU index).

[0106] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises further amino acid mutations at positions T411, D399, S400, F405, N390 or K392, such as (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, (b) D399R, D399W, D399Y or D399K, (c) S400 In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutations Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0107] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, e.g., with an amino acid modification at position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0108] In one embodiment, the heterodimerization approach described in WO2011 / 090762 is used instead, which also uses the knob-in-hole technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to Kabat EU index).

[0109] In one embodiment, the bispecific antibody or its Fc domain is of the IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is alternatively used.

[0110] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, e.g., as described in PCT Publication WO 2009 / 089004. Generally, this method involves the replacement of one or more amino acid residues at the interface of the two Fc domain subunits with a charged amino acid residue such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution at K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) and the second CH3 domain comprises an amino acid substitution at D399, E356, D356 or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution at K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain further comprises, or alternatively, an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to the Kabat EU index).

[0111] In yet further embodiments, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbering according to the Kabat EU index).

[0112] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 may alternatively be used.

[0113] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to EU index of Kabat).

[0114] As used herein in reference to bispecific anti-CCL2 antibodies, the term "wild type (WT) IgG or IgG1" refers to a bispecific antibody comprising an IgG or IgG1 heavy chain, which may include the modifications / mutations described above that promote heterodimerization, but does not contain further Fc domain modifications / mutations that increase or decrease Fc receptor binding and / or effector function as described below.

[0115] Fc domain modifications / mutations that increase or decrease Fc receptor binding and / or effector function: Modification of bispecific anti-CCL2 antibodies by sweeping techniques The bispecific anti-CCL2 antibodies were modified using a sweeping technique, enabling the bispecific anti-CCL2 antibodies to scavenge free CCl2 for extended periods of time and thus sustain their biological effects, such as anticancer effects, in vivo.

[0116] The concept of scavenging is described, for example, in Igawa et al, Immunological Reviews 270 (2016) 132-151, WO 2012 / 122011, WO 2016 / 098357 and WO 2013 / 081143, which are incorporated herein by reference.

[0117] The present invention provides a method for promoting antibody-mediated antigen uptake into cells by reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range compared to its antigen-binding activity in the neutral pH range. This promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells based on changing at least one amino acid in the antigen-binding domain of the antibody that promotes antigen uptake into cells. The present invention also provides a method for promoting antigen uptake into cells based on substituting at least one amino acid with histidine or inserting at least one histidine into the antigen-binding domain of the antibody that promotes antigen uptake into cells.

[0118] As used herein, "antigen uptake into cells" mediated by an antibody means that an antigen is taken up into cells by endocytosis. In addition, as used herein, "promoting uptake into cells" means that the intracellular uptake rate of an antibody bound to an antigen in plasma is enhanced, and / or the amount of the taken-up antigen recycled to plasma is reduced. This means that the uptake rate into cells is promoted compared to an antibody before increasing the human FcRn-binding activity of the antibody in the neutral pH range, or before increasing the human FcRn-binding activity and reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range to be lower than its antigen-binding activity in the neutral pH range. This rate is preferably improved compared to intact human IgG, more preferably compared to intact human IgG. Therefore, in the present invention, whether or not an antibody promotes antigen uptake into cells can be evaluated based on an increase in the rate of antigen uptake into cells. The rate of antigen uptake into cells can be calculated, for example, by monitoring the decrease in antigen concentration in a medium containing human FcRn-expressing cells after adding an antigen and an antibody to the medium, or by monitoring the amount of antigen uptake into human FcRn-expressing cells over time. Using the method of the present invention for promoting the rate of antibody-mediated antigen uptake into cells, for example, the rate of antigen removal from plasma can be increased by administering an antibody. Therefore, whether antibody-mediated antigen uptake into cells is promoted can also be evaluated by testing, for example, whether the rate of antigen removal from plasma is accelerated, or whether the total antigen concentration in plasma is reduced by administering an antibody.

[0119] As used herein, "total antigen concentration in plasma" refers to the sum of antibody-bound and unbound antigen concentrations, or "free antigen concentration in plasma," which is the concentration of unbound antigen. Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

[0120] As used herein, "intact human IgG" (or "wild-type (WT) human IgG)" means unmodified (except with respect to potential modifications for heterodimerization as described above) human IgG, and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "intact human IgG" as long as it is capable of binding to human FcRn in the acidic pH range. Preferably, the "intact human IgG" can be human IgG1.

[0121] The present invention also provides a method for increasing the number of antigens that a single antibody can bind to. More specifically, the present invention provides a method for increasing the number of antigens that a single antibody having human FcRn-binding activity in an acidic pH range can bind to by increasing the human FcRn-binding activity of the antibody in the neutral pH range. The present invention also provides a method for increasing the number of antigens that a single antibody having human FcRn-binding activity in the acidic pH range can bind to by changing at least one amino acid in the human FcRn-binding domain of the antibody.

[0122] The present invention provides a method for promoting antibody-mediated antigen uptake into cells. More specifically, the present invention provides a method for promoting antigen uptake into cells by an antibody having human FcRn-binding activity in an acidic pH range, the method being based on increasing the human FcRn-binding activity of the antibody in a neutral pH range. The present invention also provides a method for improving antigen uptake into cells by an antibody having human FcRn-binding activity in an acidic pH range, the method being based on changing at least one amino acid in the human FcRn-binding domain of the antibody.

[0123] The present invention also provides a method for promoting antigen uptake into cells by an antibody having human FcRn-binding activity in an acidic pH range, comprising: The present invention provides a method based on using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from those at positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the Fc domain is replaced with a different amino acid.

[0124] The present invention also provides a method for promoting antibody-mediated antigen uptake into cells by reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range compared to its antigen-binding activity in the neutral pH range. This promotes antigen uptake into cells. The present invention also provides a method for promoting antibody-mediated antigen uptake into cells based on changing at least one amino acid in the antigen-binding domain of the antibody that promotes antigen uptake into cells. The present invention also provides a method for promoting antigen uptake into cells based on substituting at least one amino acid with histidine or inserting at least one histidine into the antigen-binding domain of the antibody that promotes antigen uptake into cells.

[0125] As used herein, "antigen uptake into cells" mediated by an antibody means that an antigen is taken up into cells by endocytosis. In addition, as used herein, "promoting uptake into cells" means that the intracellular uptake rate of an antibody bound to an antigen in plasma is enhanced, and / or the amount of the taken-up antigen recycled to plasma is reduced. This means that the uptake rate into cells is promoted compared to an antibody before increasing the human FcRn-binding activity of the antibody in the neutral pH range, or before increasing the human FcRn-binding activity and reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range to be lower than its antigen-binding activity in the neutral pH range. This rate is preferably improved compared to intact human IgG, more preferably compared to intact human IgG. Therefore, in the present invention, whether or not an antibody promotes antigen uptake into cells can be evaluated based on an increase in the rate of antigen uptake into cells. The rate of antigen uptake into cells can be calculated, for example, by monitoring the decrease in antigen concentration in a medium containing human FcRn-expressing cells after adding an antigen and an antibody to the medium, or by monitoring the amount of antigen uptake into human FcRn-expressing cells over time. Using the method of the present invention for promoting the rate of antibody-mediated antigen uptake into cells, for example, the rate of antigen removal from plasma can be increased by administering an antibody. Therefore, whether antibody-mediated antigen uptake into cells is promoted can also be evaluated by testing, for example, whether the rate of antigen removal from plasma is accelerated, or whether the total antigen concentration in plasma is reduced by administering an antibody.

[0126] As used herein, "total antigen concentration in plasma" refers to the sum of antibody-bound and unbound antigen concentrations, or "free antigen concentration in plasma," which is the concentration of unbound antigen. Various methods for measuring "total antigen concentration in plasma" and "free antigen concentration in plasma" are well known in the art, as described below.

[0127] As used herein, "intact human IgG" (or "wild-type IgG") means unmodified human IgG (except with respect to potential modifications for heterodimerization as described above) and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "intact human IgG" as long as it is capable of binding to human FcRn in the acidic pH range. Preferably, the "intact human IgG" can be human IgG1.

[0128] As used herein, "parent IgG" refers to an unmodified IgG that is subsequently modified to generate a variant, so long as the modified variant of the parent IgG is capable of binding to human FcRn in the acidic pH range (hence, the parent IgG does not require binding activity to human FcRn under acidic conditions). The parent IgG may be a naturally occurring IgG, or a variant or engineered version of a naturally occurring IgG. The parent IgG may refer to the polypeptide itself, a composition comprising the parent IgG, or the amino acid sequence encoding it. It should be noted that "parent IgG" includes known commercially available recombinantly produced IgG, as outlined below. The origin of the "parent IgG" is not limited and may be obtained from any organism, non-human animal or human. Preferably, the organism is selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non-human primate. In another embodiment, the "parent IgG" may also be obtained from a cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the "parent IgG" is derived from human IgG1, but is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as the "parent IgG" as appropriate. Similarly, any class or subclass of IgG from any of the above organisms can be preferably used as the "parent IgG". Examples of variants or engineered versions of naturally occurring IgG are described in Curr Opin Biotechnol. 2009 Dec; 20(6): 685-91, Curr Opin Immunol. 2008 Aug; 20(4): 460-70, Protein Eng Des Sel. 2010 Apr; 23(4): 195-202, WO 2009 / 086320, WO 2008 / 092117, WO 2007 / 041635 and WO 2006 / 105338.

[0129] The present invention also provides a method for increasing the ability to clear plasma antigens by administering an antibody. In the present invention, the term "method for increasing the ability to clear plasma antigens" is synonymous with the term "method for enhancing the ability of an antibody to remove antigens from plasma." More specifically, the present invention provides a method for increasing the ability of an antibody having human FcRn-binding activity in an acidic pH range to remove plasma antigens by increasing the human FcRn-binding activity of the antibody in a neutral pH range. The present invention also provides a method for increasing the ability of an antibody having human FcRn-binding activity in an acidic pH range to remove plasma antigens, the method being based on changing at least one amino acid in the human FcRn-binding domain of the antibody.

[0130] The present invention also provides a parent IgG comprising a human FcRn-binding domain comprising the Fc domain of the parent IgG. The present invention provides a method for increasing the ability of an antibody having human FcRn-binding activity in the acidic pH range to exclude plasma antigens by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the Fc domain is substituted with a different amino acid.

[0131] The present invention also provides a method for increasing the ability of an antibody to clear plasma antigens by decreasing the antigen-binding activity in the acidic pH range of the antibody, which has an improved ability to clear plasma antigens compared to the antigen-binding activity in the neutral pH range. The present invention also provides a method for increasing the ability of an antibody to clear plasma antigens by changing at least one amino acid in the antigen-binding domain of the antibody with an improved ability to clear plasma antigens. The present invention also provides a method for increasing the ability of an antibody to clear plasma antigens by administering an antibody, by substituting at least one amino acid with histidine, or by inserting at least one histidine into the antigen-binding domain of the antibody with an improved ability to clear plasma antigens.

[0132] As used herein, "ability to eliminate plasma antigens" refers to the ability to remove antigens from plasma when the antibody is administered or secreted in vivo. Thus, as used herein, "increased ability of an antibody to eliminate plasma antigens" refers to an accelerated antigen removal rate from plasma upon administration of the antibody, compared to before increasing the human FcRn binding activity of the antibody in the neutral pH range, or before increasing the human FcRn binding activity and simultaneously decreasing its antigen binding activity in the acidic pH range below that in the neutral pH range. The increase in the activity of an antibody to remove antigens from plasma can be evaluated, for example, by administering a soluble antigen and an antibody in vivo and measuring the concentration of the soluble antigen in plasma after administration. If the concentration of the soluble antigen in plasma after administration of a soluble antigen and an antibody is decreased by increasing the human FcRn binding activity of the antibody in the neutral pH range, or by increasing its human FcRn binding activity and simultaneously decreasing its antigen binding activity in the acidic pH range below that in the neutral pH range, the ability of the antibody to remove plasma antigens can be determined to have increased. The form of soluble antigen can be antibody-bound or antibody-unbound antigen, the concentrations of which can be determined as "antibody-bound antigen concentration in plasma" and "antibody-unbound antigen concentration in plasma", respectively (the latter being synonymous with "free antigen concentration in plasma"). Since "total antigen concentration in plasma" means "free antigen concentration in plasma", which is the sum of antibody-bound and unbound antigen concentrations, i.e., antibody-unbound antigen concentration, the concentration of soluble antigen can be determined as "total antigen concentration in plasma". Various methods for measuring "total antigen concentration in plasma" or "free antigen concentration in plasma" are well known in the art, as described below.

[0133] The present invention also provides a method for improving the pharmacokinetics of an antibody. More specifically, the present invention provides a method for improving the pharmacokinetics of an antibody having human FcRn-binding activity in an acidic pH range by increasing the human FcRn-binding activity of the antibody in a neutral pH range. Furthermore, the present invention provides a method for improving the pharmacokinetics of an antibody having human FcRn-binding activity in an acidic pH range by changing at least one amino acid in the human FcRn-binding domain of the antibody.

[0134] The present invention also relates to a parent IgG of a human FcRn-binding domain comprising an IgG Fc domain. The present invention provides a method for improving the pharmacokinetics of an antibody having human FcRn-binding activity in the acidic pH range by using a human FcRn-binding domain comprising an amino acid sequence in which at least one amino acid selected from positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the Fc domain is substituted with a different amino acid.

[0135] The ratio of the free antigen concentration to the plasma concentration or total concentration of free antigen not bound to the antibody can be determined by methods known to those skilled in the art, for example, the method described in Pharm Res. 2006 Jan;23(1):95-103. Alternatively, if the antigen exhibits a specific function in vivo, it can be evaluated whether the antigen is bound to an antibody that neutralizes the antigen function (antagonist molecule) by testing whether the antigen function is neutralized. Whether the antigen function is neutralized can be evaluated by assaying an in vivo marker that reflects the antigen function. Whether the antigen is bound to an antibody that activates the antigen function (agonist molecule) can be evaluated by assaying an in vivo marker that reflects the antigen function.

[0136] Determination of plasma concentration of free antigen and ratio of amount of free antigen in plasma to amount of total antigen in plasma, in vivo marker assay, and such measurements are not particularly limited, but it is preferable to perform the assay after a certain time has passed after antibody administration. In the present invention, the period after antibody administration is not particularly limited, and a person skilled in the art can determine an appropriate period depending on the properties of the antibody to be administered. Such periods include, for example, 1 day after antibody administration, 3 days after antibody administration, 7 days after antibody administration, 14 days after antibody administration, and 28 days after antibody administration. In this specification, "plasma antigen concentration" means either "total antigen concentration in plasma" which is the sum of antibody-bound antigen and unbound antigen concentration, or "free antigen concentration in plasma" which is antibody-unbound antigen concentration.

[0137] The total antigen concentration in plasma can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or even more by administration of the antibody of the present invention compared to administration of a reference antibody comprising an intact human IgG Fc domain as a human FcRn-binding domain, or compared to the case where the antigen-binding domain molecule of the present invention is not administered.

[0138] In another aspect, the present invention provides bispecific anti-CCL2 antibodies that exhibit pH-dependent binding characteristics. As used herein, the term "pH-dependent binding" means that the antibody exhibits "reduced binding to CCL2 at acidic pH compared to binding at neutral pH" (for the purposes of this disclosure, both terms may be used interchangeably). For example, an antibody "having pH-dependent binding properties" includes an antibody that binds to CCL2 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the bispecific antibodies of the present invention bind to CCL2 with at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold or more affinity at neutral pH than at acidic pH. In some embodiments, the antibody binds to CCL2 with higher affinity at pH 7.4 than at pH 5.8. In further embodiments, the antibody binds to CCL2 with at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold or more higher affinity at pH 7.4 than at pH 5.8.

[0139] When an antigen is a soluble protein, the antibody can have a longer plasma half-life than the antigen itself and can act as a carrier of the antigen, so that binding of the antibody to the antigen can result in an increase in the half-life of the antigen in plasma (i.e., a decrease in the clearance of the antigen from plasma). This is due to recycling of the antigen-antibody complex by FcRn via the endosomal pathway in cells (Roopenian, Nat. Rev. Immunol. 7(9):715-725(2007)). However, antibodies with pH-dependent binding properties that bind their antigens in the neutral extracellular environment and release the antigen into acidic endosomal compartments after entry into the cell are expected to have superior properties in terms of antigen neutralization and clearance compared to their pH-independent binding counterparts (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009 / 125825).

[0140] The "affinity" of an antibody for CCL2, for the purposes of this disclosure, is expressed in terms of the antibody's KD. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody that binds to that antigen, the weaker its binding affinity for that particular antigen. Thus, as used herein, the phrase "higher affinity at neutral pH than at acidic pH" "pH-dependent binding" means that the KD of an antibody that binds to CCL2 at acidic pH is higher than the KD of an antibody that binds to CCL2 at neutral pH. For example, in the context of the present invention, an antibody is considered to bind to CCL2 with higher affinity at neutral pH than at acidic pH if the KD of an antibody that binds to CCL2 at acidic pH is at least two-fold higher than the KD of an antibody that binds to CCL2 at neutral pH. Thus, the invention includes antibodies that bind to CCL2 at acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more greater than the KD of antibodies that bind to CCL2 at neutral pH. In another embodiment, the KD value of the antibody at neutral pH can be 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less. In another embodiment, the KD value of the antibody at acidic pH can be 10-9M, 10-8M, 10-7M, 10-6M, or more.

[0141] In further embodiments, an antibody is considered to bind with higher affinity at neutral pH than at acidic pH if the KD of the antibody that binds to CCL2 at pH 5.8 is at least 2-fold greater than the KD of the antibody that binds to CCL2 at pH 7.4. In some embodiments, provided antibodies bind to CCL2 at pH 5.8 with a KD that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times greater than the KD of the antibody that binds to CCL2 at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 can be 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10-9M, 10-8M, 10-7M, 10-6M or more.

[0142] The binding properties of an antibody to a particular antigen can also be expressed in terms of the antibody's kd. The kd of an antibody refers to the dissociation rate constant of an antibody to a particular antigen and is expressed in reciprocal seconds (i.e., sec-1). An increase in the kd value means that the antibody binds weaker to that antigen. Thus, the present invention includes antibodies that bind to CCL2 at acidic pH with a higher kd value than at neutral pH. The present invention includes antibodies that bind to CCL2 at acidic pH with a Kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more, greater than the Kd of an antibody that binds to CCL2 at neutral pH. In another embodiment, the kd value of the antibody at neutral pH can be 10-2 1 / s, 10-3 1 / s, 10-4 1 / s, 10-5 1 / s, 10-6 1 / s or less. In another embodiment, the kd value of the antibody at acidic pH can be 10-3 1 / s, 10-2 1 / s, 10-1 1 / s or more. The invention also includes antibodies that bind to CCL2 with a higher kd value at pH 5.8 than at pH 7.4. The invention includes antibodies that bind to CCL2 at pH 5.8 with a Kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more greater than the Kd of the antibody that binds to CCL2 at pH 7.4. In another embodiment, the antibody's kd value at pH 7.4 can be 10-2 1 / s, 10-3 1 / s, 10-4 1 / s, 10-5 1 / s, 10-6 1 / s or less. In another embodiment, the antibody's kd value at pH 5.8 can be 10-3 1 / s, 10-2 1 / s, 10-1 1 / s or more.

[0143] In certain cases, "reduced binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed in terms of the ratio of the KD value of an antibody that binds to CCL2 at acidic pH to the KD value of an antibody that binds to CCL2 at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral KD ratio of 2 or more, for purposes of the present invention, the antibody may be considered to exhibit "reduced binding to CCL2 at acidic pH compared to binding at neutral pH". In certain embodiments, the pH5.8 / pH7.4 KD ratio for an anti-CCL2 antibody of the present invention is 2 or more. In certain exemplary embodiments, the acidic / neutral KD ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of an antibody at neutral pH can be 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less. In another embodiment, the KD value of an antibody at acidic pH can be 10-9M, 10-8M, 10-7M, 10-6M or more. In a further example, an antibody can be considered to exhibit "decreased binding to CCL2 at acidic pH compared to binding at neutral pH" if the antibody exhibits a KD ratio pH5.8 / pH7.4 of 2 or greater. In certain exemplary embodiments, the antibody's pH 5.8 / pH 7.4 KD ratio can be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the antibody's KD value at pH 7.4 can be 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M or less. In another embodiment, the antibody's KD value at pH 5.8 can be 10-9M, 10-8M, 10-7M, 10-6M or more.

[0144] In certain cases, "reduced binding to CCL2 at acidic pH compared to binding at neutral pH" is expressed in terms of the ratio of the Kd value of the antibody that binds to CCL2 at acidic pH to the Kd value of the antibody that binds to CCL2 at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral Kd ratio of 2 or greater, then for purposes of the present invention, the antibody may be considered to exhibit "reduced binding to CCL2 at acidic pH compared to binding at neutral pH". In certain exemplary embodiments, the pH5.8 / pH7.4 kd ratio of the antibody of the present invention is 2 or greater. In certain exemplary embodiments, the acidic / neutral Kd ratio for the antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. In another embodiment, the kd value of the antibody at neutral pH can be 10-2 1 / s, 10-3 1 / s, 10-4 1 / s, 10-5 1 / s, 10-6 1 / s or less. In another embodiment, the kd value of the antibody at acidic pH can be 10-3 1 / s, 10-2 1 / s, 10-1 1 / s or more. In certain exemplary embodiments, the pH5.8 / pH7.4 kd ratio of the antibodies of the invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. In another embodiment, the antibody's kd value at pH 7.4 can be 10-2 1 / s, 10-3 1 / s, 10-4 1 / s, 10-5 1 / s, 10-6 1 / s or less. In another embodiment, the antibody's kd value at pH 5.8 can be 10-3 1 / s, 10-2 1 / s, 10-1 1 / s or more.

[0145] As used herein, the term "acidic pH" refers to a pH between 4.0 and 6.5. The term "acidic pH" includes any one of the following pH values: 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In a particular embodiment, the "acidic pH" is 5.8.

[0146] As used herein, the term "neutral pH" refers to a pH between 6.7 and about 10.0. The term "neutral pH" includes any one of the following pH values: 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In a particular embodiment, the "neutral pH" is 7.4.

[0147] The KD and kd values ​​expressed herein can be determined using a surface plasmon resonance-based biosensor to characterize the antibody-antigen interaction. The KD and kd values ​​can be determined at 25°C or 37°C.

[0148] In a further aspect, the present invention provides bispecific anti-CCL2 antibodies that form immune complexes (i.e., antigen-antibody complexes) with CCL2. In certain embodiments, two or more bispecific anti-CCL2 antibodies bind to two or more CCL2 molecules to form immune complexes. This is possible because the antibodies have two antigen-binding sites, while CCL2 exists as a homodimer comprising two CCL2 molecules.

[0149] Generally speaking, when two or more antibodies form an immune complex with two or more antigens, the resulting immune complex can strongly bind to Fc receptors present on the cell surface due to the avidity effect via the Fc regions of the antibodies in the complex, and can then be internalized into the cell with high efficiency. Thus, the above-mentioned anti-CCL2 antibodies capable of forming an immune complex containing two or more anti-CCL2 antibodies and two or more CCL2 molecules can bring about rapid clearance of CCL2 from plasma in vivo through strong binding to Fc receptors due to the avidity effect.

[0150] Furthermore, antibodies with pH-dependent binding properties are believed to have superior properties in terms of antigen neutralization and clearance compared to their pH-independent binding counterparts (Igawa et al., Nature Biotech. 28(11):1203-1207(2010); Devanaboyina et al. mAbs 5(6):851-859(2013); WO 2009 / 125825). Therefore, antibodies with both of the above properties, i.e., antibodies that have pH-dependent binding properties and form immune complexes containing two or more antigens and two or more antibodies, are expected to have superior properties of highly promoting the removal of antigens from plasma (WO 2013 / 081143).

[0151] In one aspect, the invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity comprising at least two amino acid changes including: (a) one amino acid change at position 236, and (b) at least one amino acid change at at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396 (according to EU numbering).

[0152] In one aspect, the invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity comprising an amino acid change at position 236 (EU numbering).

[0153] In one aspect, the invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity comprising at least two amino acid changes including: (a) one amino acid change at position 236, and (b) at least one amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering). In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396 (according to EU numbering). In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 268, 295, 326, and 330 (according to EU numbering).

[0154] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having enhanced Fc gamma RIIb binding activity, the variant Fc region comprising any one of the following amino acid changes (1) to (37): (1) at positions 231, 236, 239, 268, and 330; (2) at positions 231, 236, 239, 268, 295, and 330; (3) at positions 231, 236, 268, and 330; (4) at positions 231, 236, 268, 295, and 330; (5) at positions 232, 236, 239, 268, 295, and 330; (6) at positions 232, 236, 268, 295, and 330. 0;(7) positions 232, 236, 268 and 330;(8) positions 235, 236, 268, 295, 326 and 330;(9) positions 235, 236, 268, 295 and 330;(10) positions 235, 236, 268 and 330;(11) positions 235, 236, 268, 330 and 396;(12) positions 235, 236, 268 and 396;(13) positions 236, 239, 268, 295, 298 and 330;(14) positions 236, 239, 268, 295, 326 and 330;(15) positions 236, 239, 268, 295 and 330;( 16) positions 236, 239, 268, 298 and 330; (17) positions 236, 239, 268, 326 and 330; (18) positions 236, 239, 268 and 330; (19) positions 236, 239, 268, 330 and 396; (20) positions 236, 239, 268 and 396; (21) positions 236 and 268; (22) positions 236, 268 and 295; (23) positions 236, 268, 295, 298 and 330; (24) positions 236, 268, 295, 326 and 330; (25) positions 236, 268, 295, 326, 330 and 39 6;(26) positions 236, 268, 295 and 330;(27) positions 236, 268, 295, 330 and 396;(28) positions 236, 268, 298 and 330;(29) positions 236, 268, 298 and 396;(30) positions 236, 268, 326 and 330;(31) positions 236, 268, 326, 330 and 396;(32) positions 236, 268 and 330;(33) positions 236, 268, 330 and 396;(34) positions 236, 268 and 396;(35) positions 236 and 295;(36) positions 236, 330 and 396;and (37) positions 236 and 396 (according to EU numbering);

[0155] In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises at least one amino acid selected from the group consisting of: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, Ile at position 232; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (h) Glu, Ile, Met, Gln, Tyr at position 238. (i) Ile, Leu, Asn, Pro, Val at position 239; (j) Ile at position 264; (k) Phe at position 266; (l) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position 295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325; (r) Thr at position 326; (s) Ala, His, Leu at position 267; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396 (according to EU numbering).

[0156] In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises at least one amino acid change (e.g., substitution) selected from the group consisting of: (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; (k) Lys, Met at position 396 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Thr at position 236, Asp at position 268, and Lys at position 330 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 (according to EU numbering). In a further embodiment, the variant Fc region with enhanced Fc gamma RIIb binding activity comprises the following amino acid changes (e.g., substitutions): Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330 (according to EU numbering).

[0157] In another aspect, the invention provides isolated polypeptides comprising a variant Fc region with an increased isoelectric point (pI). In certain embodiments, the variant Fc region described herein comprises at least two amino acid changes to the parent Fc region. In certain embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region compared to the isoelectric point (pI) of the parent Fc region. They are based on the discovery that antibodies with an increased pI due to modification of at least two amino acid residues can facilitate antigen clearance from plasma, for example, when the antibody is administered in vivo.

[0158] In the present invention, pI may be either a theoretically determined pI or an experimentally determined pI. The value of pI may be determined, for example, by isoelectric focusing known to those skilled in the art. The theoretical pI may be calculated, for example, using gene / amino acid sequence analysis software (such as Genetyx).

[0159] In one embodiment, the pI value may be increased by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, and at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more compared to before modification.

[0160] In certain embodiments, amino acids for increasing pI can be exposed on the surface of the variant Fc region. In the present invention, an amino acid that can be exposed on the surface generally refers to an amino acid residue located on the surface of a polypeptide constituting a variant Fc region. An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can contact a solvent molecule (generally mostly water molecules). However, not all side chains are necessarily in contact with a solvent molecule, and an amino acid is defined as an "amino acid residue located on the surface" if it is in contact with a solvent molecule even partially. An amino acid residue located on the surface of a polypeptide also includes an amino acid residue located near the surface, whereby a side chain may have a charge influence from another amino acid residue that is in contact with a solvent molecule, even if only partially. A person skilled in the art can prepare a homology model of a polypeptide, for example, using commercially available software. Alternatively, it is possible to use a method known to a person skilled in the art, such as X-ray crystallography. An amino acid residue that can be exposed on the surface is determined, for example, using coordinates from a three-dimensional model using a computer program such as the InsightII program (Accelrys). The surface-exposable sites can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55: 379-400 (1971)); Connolly (J. Appl. Cryst. 16: 548-558 (1983)). The surface-exposable sites can be determined using software suitable for protein modeling and three-dimensional structural information. Software available for such purposes includes, for example, SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires a user-input size parameter, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms or less. Furthermore, a method for determining surface-exposable regions using software for personal computers has been described by Pacios (Comput. Chem. 18 (4): 377-386 (1994); J. Mol. Model. 1: 46-53 (1995)).Based on such information, appropriate amino acid residues to be located on the surface of the polypeptide constituting the variant Fc region can be selected.

[0161] In certain embodiments, the polypeptide comprises both a variant Fc region and an antigen binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in a subject's bodily fluids (e.g., plasma, interstitial fluid, lymphatic fluid, peritoneal fluid, and pleural fluid). The antigen may be a membrane antigen.

[0162] In a further embodiment, the antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) and metal ion concentration. In this specification, metal ions refer to ions of group I elements excluding hydrogen, such as alkali metals and copper group elements, group II elements such as alkaline earth metals and zinc group elements, group III elements excluding boron, group IV elements excluding carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI and VII, and metal elements such as antimony, bismuth, and polonium. In the present invention, metal ions include calcium ions, for example, as described in WO2012 / 073992 and WO2013 / 125667. In one embodiment, the "ion concentration conditions" may be conditions that focus on the difference in biological behavior of the antigen-binding domain between low ion concentration and high ion concentration. Furthermore, "the antigen-binding activity of the antigen-binding domain changes depending on ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (such antigen-binding domains are referred to herein as "ion concentration-dependent antigen-binding domains"). The antigen-binding activity of the antigen-binding domain under high ion concentration conditions can be higher (stronger) and lower (weaker) than under low ion concentration conditions. In one embodiment, the ion concentration-dependent antigen-binding domain (such as a pH-dependent antigen-binding domain or a calcium ion concentration-dependent antigen-binding domain) can be obtained by known methods, for example, the methods described in WO2009 / 125825, WO2012 / 073992 and WO2013 / 046722.

[0163] In the present invention, the antigen-binding activity of the antigen-binding domain under high calcium ion concentration conditions may be higher than that under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but is preferably close to the calcium ion plasma (blood) concentration in vivo, and may be a concentration selected from 100 microM to 10 mM, 200 microM to 5 mM, 400 microM to 3 mM, 200 microM to 2 mM, 400 microM to 1 mM, and 500 microM to 2.5 mM. On the other hand, the low calcium ion concentration is not particularly limited, but is preferably close to the calcium ion concentration in an early endosome in vivo, and may be a concentration selected from 0.1 microM to 30 microM, 0.2 microM to 20 microM, 0.5 microM to 10 microM, 1 microM to 5 microM, or 2 microM to 4 microM.

[0164] In one embodiment, the ratio of antigen-binding activity under low calcium ion concentration conditions to that under high calcium ion concentration conditions is not limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, i.e., KD (low calcium ion concentration conditions) / KD (high calcium ion concentration conditions), is 2 or more, 10 or more, and 40 or more. The upper limit of this ratio may be 400, 1000, and 10000, as long as such an antigen-binding domain can be produced by a technique known to those skilled in the art. In addition, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under low calcium ion concentration conditions to kd under high calcium ion concentration conditions, i.e., kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions), is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of this ratio may be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the technical common sense of those skilled in the art.

[0165] In the present invention, the antigen-binding activity of the antigen-binding domain under a low hydrogen ion concentration (neutral pH) may be higher than that under a high hydrogen ion concentration (acidic pH). The acidic pH is preferably close to the in vivo pH in the early endosome, and may be, for example, a pH selected from pH 4.0 to pH 6.5, a pH selected from pH 4.5 to pH 6.5, a pH selected from pH 5.0 to pH 6.5, and a pH selected from pH 5.5 to pH 6.5. The acidic pH may be, for example, pH 5.8 and pH 6.0. In a specific embodiment, the acidic pH is pH 5.8. The neutral pH may be, for example, a pH selected from pH 6.7 to pH 10.0, a pH selected from pH 6.7 to pH 9.5, a pH selected from pH 7.0 to pH 9.0, or a pH selected from pH 7.0 to pH 8.0, and is preferably close to the in vivo pH in plasma (blood). The neutral pH may be, for example, pH 7.4 and pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

[0166] In one embodiment, the ratio of antigen-binding activity under acidic pH conditions to that under neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or more, 10 or more, and 40 or more. The upper limit of this ratio may be 400, 1000, and 10000, as long as such an antigen-binding domain can be produced by a technique known to those skilled in the art. In addition, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions to kd under neutral pH conditions, i.e., kd(acidic pH conditions) / kd(neutral pH conditions), is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio may be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the technical common sense of those skilled in the art.

[0167] In one embodiment, as described in, for example, WO 2009 / 125825, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0 to 8.0, and / or at least one amino acid having a side chain pKa of 4.0 to 8.0 is inserted into the antigen-binding domain. The amino acid may be substituted and / or inserted at any site, as long as the antigen-binding activity of the antigen-binding domain is weaker under acidic pH conditions than under neutral pH conditions compared to before the substitution or insertion. When the antigen-binding domain has a variable region and CDR, the site may be within the variable region and CDR. The number of amino acids to be substituted or inserted can be appropriately determined by one skilled in the art, and this number may be 1 or more. The antigen-binding activity of the antigen-binding domain can be changed according to hydrogen ion concentration conditions using an amino acid having a side chain pKa of 4.0 to 8.0. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), as well as unnatural amino acids such as histidine analogs (US Patent Application Publication No. 2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). Amino acids with side chain pKas of 6.0 to 7.0 can also be used, such as His (H).

[0168] In another embodiment, preferred antigen binding domains of variant Fc regions with increased pI are described and can be obtained by the methods described in WO2016 / 125495 and WO2017 / 046994.

[0169] In certain embodiments, the variant Fc region with increased pi comprises at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, 431 (according to EU numbering).

[0170] In a further embodiment, the variant Fc region with increased pi comprises at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, 413 (according to EU numbering).

[0171] In another aspect, the present invention provides a polypeptide comprising a variant Fc region having an increased pI, comprising any one of the following amino acid changes (1) to (10): (1) positions 311 and 341; (2) positions 311, 343; (3) positions 311, 343, 413; (4) positions 311, 384, 413; (5) positions 311, 399; (6) positions 311, 401; (7) positions 311, 413; (8) positions 400, 413; (9) positions 401, 413; and (10) positions 402 and 413 (according to EU numbering).

[0172] In one aspect, the present invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity and increased pI, comprising at least three amino acid changes, including: (a) at least one amino acid change at at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 40 26, 327, 328, 330, 331, 332, 334, and 396 (according to EU numbering); and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, 431 (according to EU numbering).

[0173] In one aspect, the invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity and increased pI comprising at least three amino acid changes including: (a) at least one amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330 and 396 (according to EU numbering); and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, 413 (according to EU numbering).

[0174] In another aspect, the present invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity and increased pI, comprising any one of the following amino acid changes (1) at positions 235, 236, 268, 295, 311, 326, 330, and 343; (2) at positions 236, 268, 295, 311, 326, 330, and 343; (3) at positions 236, 268, 295, 311, 330, and 41 3;(4) positions 236, 268, 311, 330, 396 and 399;(5) positions 236, 268, 311, 330 and 343;(6) positions 236, 268, 311, 330, 343 and 413;(7) positions 236, 268, 311, 330, 384 and 413;(8) positions 236, 268, 311, 330 and 413; and(9) positions 236, 268, 330, 396, 400 and 413 (according to EU numbering).

[0175] In one aspect, the invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity and increased pI comprising at least three amino acid changes including: (a) at least one amino acid change at at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330; and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, 431 (according to EU numbering). In a further embodiment, the polypeptide comprises at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, 413 (according to EU numbering).

[0176] In another aspect, the present invention provides a polypeptide comprising a variant Fc region with enhanced Fc gamma RIIb binding activity and increased pI, comprising any one of the following amino acid changes (1) at positions 234, 238, 250, 264, 307, 311, 330, and 343; (2) at positions 234, 238, 250, 264, 307, 311, 330, and 413; (3) at positions 234, 238, 250, 264, 307, 311, 330, and 413; 234, 238, 250, 264, 267, 307, 311, 330 and 343;(4) positions 234, 238, 250, 264, 267, 307, 311, 330 and 413;(5) positions 234, 238, 250, 267, 307, 311, 330 and 343;(6) positions 234, 238, 250, 267, 307, 311, 330 and 413;(7) positions 234, 238, 250, 3 07, 311, 330 and 343;(8) positions 234, 238, 250, 307, 311, 330 and 413;(9) positions 238, 250, 264, 267, 307, 311, 330 and 343;(10) positions 238, 250, 264, 267, 307, 311, 330 and 413;(11) positions 238, 250, 264, 307, 311, 330 and 343;(12) positions 238, 250, 264, 307, 311, 330 and 413; (13) positions 238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307, 311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and (16) positions 238, 250, 307, 311, 330 and 413 (according to EU numbering).

[0177] Moreover, amino acid changes made for other purposes can be combined in the variant Fc regions described herein. For example, amino acid substitutions that improve FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356(2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524(2006); Petkova et al., Intl. Immunol. 18(12):1759-1769(2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159(2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320) and amino acid substitutions to improve antibody heterogeneity or stability (WO 2009 / 041613) may be made. Alternatively, polypeptides with antigen clearance promoting properties as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704 and WO2013 / 180201, polypeptides with specific binding properties to target tissues as described in WO2013 / 180200, polypeptides with repetitive binding properties to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992 and WO2013 / 047752 can be combined with the variant Fc regions described herein. Alternatively, amino acid changes disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the variant Fc regions described herein to confer binding capacity to other antigens. Alternatively, the variant Fc regions described herein may be combined with amino acid changes that decrease the pI of the constant regions (WO 2012 / 016227) to increase plasma retention, or with amino acid changes that increase the pI of the constant regions (WO 2014 / 145159) to enhance cellular uptake.Alternatively, amino acid changes that increase the pI of the constant region (WO 2016 / 125495) can be combined in the variant Fc region described herein to facilitate clearance of the target molecule from plasma. In one embodiment, such changes can include, for example, a substitution at at least one position selected from the group consisting of 311, 343, 384, 399, 400 and 413 according to EU numbering. In a further embodiment, such substitutions can be the replacement of the amino acid at each position with Lys and Arg.

[0178] Amino acid changes that enhance human FcRn binding activity under acidic pH can also be combined into the variant Fc regions described herein. Specifically, such changes include, for example, substitutions of Leu with Met at position 428 and Ser with Asn at position 434 according to the EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); substitution of Ala with Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); substitution of Tyr with Met at position 252, substitution of Thr with Ser at position 254, and substitution of Glu with Thr at position 256 (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)); substitution of Gln with Thr at position 250 and substitution of Leu with Met at position 428 (Hinton et al., J. Biol. Chem. 281:23514-23524 (2006)). al., J. Immunol. 176(1):346-356 (2006); substitution of His with Asn at position 434 (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), as well as WO 2010 / 106180, WO 2010 / 045193, WO 2009 / 058492, WO 2008 / 022152, WO 2006 / 050166, WO 2006 / 053301, WO 2006 / 031370, WO 2005 / 123780, WO 2005 / 047327, WO 2005 / 037867, WO 2005 / 037868, WO 2005 / 037869 ... The alterations may include those described in WO 2004 / 035752 or WO 2002 / 060919. Such alterations may include, for example, at least one alteration selected from the group consisting of a substitution of Met with Leu at position 428, a substitution of Asn with Ala at position 434, and a substitution of Tyr with Thr at position 436. These alterations may further include a substitution of Arg for Gln at position 438 and / or a substitution of Glu for Ser at position 440 (WO 2016 / 125495).

[0179] Exemplary Bispecific Anti-CCL2 Antibodies One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, The bispecific antibody a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Including, A) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or B) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or C) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or D) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or E) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or F) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or G) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or H) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or I) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or J) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or K) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or L) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or M) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO:94; or N) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or O) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or P) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; A bispecific antibody, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.

[0180] One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, Bispecific antibodies a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Including, i) the first antigen-binding site is (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 57 (wherein X is I), (b) CDR-H2 comprising the amino acid sequence GX of SEQ ID NO: 58 1 IX 2 IFX 3 TANYAQKFQG (where X 1 is V and X 2 is P and X 3 (c) a CDR-H2 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60, (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 62. Including, ii) the second antigen-binding site comprises: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 76; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77 (wherein X is D); and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 78 (wherein X is E); (d) the amino acid sequence KAX of SEQ ID NO: 79 1 EDIYNRX 2 A (where X 1 is F and X 2 (e) a CDR-L2 comprising the amino acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, wherein X is R. The bispecific antibody comprises:

[0181] One embodiment of the present invention is a bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, Bispecific antibodies a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Including, i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; A bispecific antibody, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.

[0182] In one embodiment, L1 and L2 are polypeptide linkers comprising the amino acids glycine and serine, where the repeated glycines are limited to a maximum of four consecutive glycines and no serine is directly linked to another serine.

[0183] In one embodiment, L1 is a polypeptide linker having a length of 9-11 amino acids and L2 is a polypeptide linker having a length of 9-11 amino acids.

[0184] In one embodiment, L1 and L2 are polypeptide linkers selected from the group of GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185), GGSGGSGGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), and GGGGSGGSGG (SEQ ID NO: 191).

[0185] In one embodiment, L1 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188) and L2 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188).

[0186] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG isotype, preferably the human IgG1 isotype.

[0187] In one embodiment, the bispecific antibodies described herein are not cross-reactive to other human CCL homologues, and in particular exhibit 100-fold lower binding to other CCL homologues (selected from the group of CCL8, CCL7 and CCL13) compared to binding to CCL2.

[0188] In one embodiment, the bispecific antibodies described herein bind to a first and a second epitope on human CCL2 in an ion-dependent manner.

[0189] In one embodiment, the bispecific antibodies described herein bind to human CCL2 in a pH-dependent manner, and both the first antigen-binding site and the second antigen-binding site bind to CCL2 with higher affinity at neutral pH than at acidic pH.

[0190] In one embodiment, the bispecific antibodies described herein bind to human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

[0191] In one embodiment, the in vivo clearance rate of human CCL2 (ml / day / kg) after administration of a bispecific antibody comprising a constant heavy domain of the human wild type IgG1 isotype (or an Fc domain thereof) is at least 15-fold higher, in particular at least 20-fold higher, compared to the in vivo clearance rate of human CCL2 (ml / day / kg) after administration of a bispecific antibody comprising an Fc gamma receptor silencing constant heavy domain of the human IgG1 isotype (or an Fc domain thereof) comprising the mutations L234A, L235A, P329G (Kabat EU numbering), when a single dose of 10 ml / kg of preformed immune complexes consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg human CCL2 is administered to FcRn transgenic mice.

[0192] In one embodiment, the in vivo clearance rate (ml / day / kg) of human CCL2 following administration of a bispecific antibody comprising a constant heavy domain of the human wild type IgG1 isotype (or an Fc domain thereof) is at least 2-fold higher (in one embodiment, at least 5-fold higher, in one embodiment, at least 10-fold higher, in one embodiment, at least 20-fold higher) compared to the in vivo clearance rate (ml / day / kg) of human CCL2 following administration of a bispecific antibody comprising an Fc gamma receptor silencing constant heavy domain of the human IgG1 isotype (or an Fc domain thereof) comprising the mutations L234A, L235A, P329G (Kabat EU numbering) when a single dose of 10 ml / kg of preformed immune complexes consisting of 20 mg / kg of each bispecific antibody and 0.1 mg / kg human CCL2 is administered to FcRn transgenic mice.

[0193] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0194] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L235W, G236N, H268D, Q295L, K326T and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) N434A (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0195] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) N434A (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0196] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) N434A (suitable for increasing affinity to FcRn for longer plasma half-life), and iii) Contains one or more of Q438R and S440E (suitable for inhibiting rheumatoid factor binding).

[0197] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): Contains one or more of Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

[0198] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L234Y, P238D, T250V, V264I, T307P and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0199] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0200] In one embodiment, the constant heavy chain domains CH1, hinge, CH2 and CH3 are of the human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) N434A and (suitable for increasing affinity for FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) Contains one or more of the following:

[0201] In one embodiment, such a bispecific antibody comprises (independently and in addition to the mutations listed above) the following mutations (Kabat EU numbering): i) S354C and T366W in one of the heavy chain constant CH3 domains ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant CH3 domain Includes.

[0202] In one embodiment, other heterodimerization-promoting mutations described above in the section on heterodimerization-promoting Fc domain modifications can be used in place of the exemplary knobs to the hole modifications above.

[0203] A particular embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 175 and a polypeptide comprising an amino acid sequence at least 98% or 99% identical to the sequence of SEQ ID NO: 176.

[0204] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, the bispecific antibody comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 175 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 176.

[0205] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 177 and a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 178.

[0206] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, the bispecific antibody comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 177 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 178.

[0207] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 179 and a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 180.

[0208] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, the bispecific antibody comprising a polypeptide comprising the amino acid sequence of sequence SEQ ID NO: 179 and a polypeptide comprising the amino acid sequence of sequence SEQ ID NO: 180.

[0209] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 181 and a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID NO: 182.

[0210] A specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site which (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site which (specifically) binds to a second (different) epitope on human CCL2, the bispecific antibody comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 181 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 182.

[0211] Recombinant methods and compositions Antibodies may be produced, for example, using recombinant methods and compositions described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-CCL2 antibody (either bispecific or monospecific) described herein. Such a nucleic acid may encode an amino acid sequence comprising one or all of the VL and / or one or all of the VH (e.g., the light and / or heavy chains of the antibody) of a monospecific or bispecific antibody. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, a HEK293 cell, or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-CCL2 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, such as, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0212] For recombinant production of anti-CCL2 cells, such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0213] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibodies of the invention can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0214] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414, and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0215] Also suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0216] Plant cell cultures can also be used as hosts, see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0217] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells, as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, e.g., Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, e.g., Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0218] In another aspect, the invention is based, in part, on the discovery that the modified monospecific antibodies described herein exhibit improved pH-dependent binding properties and are therefore particularly useful in generating the bispecific antibodies of the invention.

[0219] Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the bispecific anti-CCL2 antibodies provided herein are useful for detecting the presence of CCL2 in a biological sample. As used herein, the term "detect" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as immune cells, or T cell infiltrates and or tumor cells.

[0220] In one embodiment, a bispecific anti-CCL2 antibody is provided for use in methods of diagnosis and detection. In a further aspect, a method of detecting the presence of CCL2 in a biological sample is provided. In a particular embodiment, the method comprises contacting a biological sample with a bispecific anti-CCL2 antibody as described herein under conditions permissive for binding of the bispecific anti-CCL2 antibody to CCL2, and detecting whether a complex is formed between the bispecific anti-CCL2 antibody and CCL2. Such a method may be an in vitro or in vivo method. In one embodiment, the bispecific anti-CCL2 antibody is used to select subjects eligible for treatment with the bispecific anti-CCL2 antibody (e.g., CCL2 is a biomarker for selecting patients).

[0221] In certain embodiments, a labeled bispecific anti-CCL2 antibody is provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron dense, chemiluminescent, and radioactive labels) and moieties that are indirectly detected, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131Examples of suitable oxidases include, but are not limited to, I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0222] Pharmaceutical preparations Pharmaceutical formulations of the bispecific anti-CCL2 antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired degree of purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine. Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0223] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine acetate buffer.

[0224] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide additional. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0225] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0226] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0227] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0228] Therapeutic Methods and Compositions Any of the bispecific anti-CCL2 antibodies provided herein can be used in therapeutic methods.

[0229] In one aspect, a bispecific anti-CCL2 antibody is provided for use as a medicament. In a further aspect, a bispecific anti-CCL2 antibody and use in the treatment of cancer are provided. In a particular embodiment, a bispecific anti-CCL2 antibody is provided for use in a method of treatment. In a particular embodiment, the invention provides a bispecific anti-CCL2 antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific anti-CCL2 antibody.

[0230] In a further embodiment, the invention provides that bispecific anti-CCL2 antibodies inhibit immune suppression in tumors, thus sensitizing tumors to immune stimulatory genes such as anti-PD1, anti-PDL-1 antagonists, etc.

[0231] Thus, one aspect thereof is the combination of the bispecific anti-CCL2 antibodies described herein with cancer immunotherapies such as anti-PD1, anti-PDL-1 antagonists.

[0232] As used herein, the term "cancer" refers to any cancer, including, for example, lung cancer, non-small cell lung (NSCL) cancer, lung bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, and the like. cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory aspects of any of the above cancers), or a combination of one or more of the above cancers.

[0233] The "individual" according to any of the above embodiments is preferably a human. In a further aspect, the invention provides the use of a bispecific anti-CCL2 antibody in the manufacture and preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer. In a further embodiment, the medicament is for use in a method of treating cancer, the method comprising administering an effective amount of the medicament to an individual having cancer. In a further embodiment, the medicament is for inducing cell-mediated lysis of cancer cells. In a further embodiment, the medicament is for use in a method of inducing cell-mediated lysis of cancer cells in an individual suffering from cancer, the method comprising administering to the individual an amount of the medicament effective to induce apoptosis in the cancer cells / or to inhibit cancer cell proliferation. The "individual" according to any of the above embodiments may be a human.

[0234] In a further aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a bispecific anti-CCL2 antibody. The "individual" according to any of the above embodiments may be a human.

[0235] In a further aspect, the invention provides a method of inducing cell-mediated lysis of cancer cells in an individual suffering from cancer. In one embodiment, the method comprises administering to the individual an effective amount of a bispecific anti-CCL2 antibody to induce cell-mediated lysis of cancer cells in the individual suffering from cancer. In one embodiment, the "individual" is a human.

[0236] In another aspect of the invention, bispecific anti-CCL2 antibodies are provided for use in the treatment of inflammatory and autoimmune diseases. In a particular embodiment, the invention provides a bispecific anti-CCL2 antibody for use in a method of treating an individual having an inflammatory or autoimmune disease comprising administering to the individual an effective amount of the bispecific anti-CCL2 antibody.

[0237] In some embodiments, the inflammatory and autoimmune diseases are autoimmune disorders, inflammatory disorders, fibrotic disorders, granulocytic (neutrophilic and eosinophilic), monocytic, or lymphocytic disorders, and disorders associated with an increase in the number or distribution of normal or abnormal tissue-resident cells (such as mast cells, macrophages, and lymphocytes) or interstitial cells (such as fibroblasts, myofibroblasts, smooth muscle cells, epithelium, and endothelium). In some embodiments, the disorder is a pulmonary disorder. In some embodiments, the pulmonary disorder includes those associated with granulocytic (eosinophilic and / or neutrophilic) pulmonary inflammation, infection-induced pulmonary conditions (viral (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus), bacterial, or fungal (e.g., Aspergillus) triggers. In some embodiments, the disorder includes allergen-induced pulmonary conditions, toxic environmental pollutant-induced pulmonary conditions (e.g., asbestosis, silicosis, and beryllium disease), gastric aspiration-induced pulmonary conditions, and immune dysregulation or cystic fibrosis. Associated with inflammatory conditions with genetic predisposition. In some embodiments, the disorder is physical trauma-induced pulmonary conditions (e.g., ventilator injury), emphysema, cigarette-induced emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), airway exacerbation, and acute respiratory distress syndrome (ARDS). In some embodiments, the inflammatory lung disorder is COPD.

[0238] In some embodiments, the inflammatory lung disorder is asthma. In some embodiments, the asthma is persistent, chronic, severe asthma with acute events that worsen symptoms (exacerbations or flare-ups) that can be life-threatening. In some embodiments, the asthma is atopic (also known as allergic) asthma, non-allergic asthma (e.g., often caused by infection with respiratory viruses (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus) or inhaled irritants (air pollutants, smog, diesel particulates, volatile chemicals and gases, indoors or outdoors, or even by cold, dry air).

[0239] In some embodiments, the asthma is intermittent or exercise-induced asthma due to acute or chronic primary or secondary exposure to "smoke" (typically cigarettes, cigars, pipes), inhalation, or smoking (tobacco, marijuana, or other such substances), or asthma caused by new intake of aspirin or related NSAIDs. In some embodiments, the asthma is mild asthma or corticosteroid-naïve asthma, newly diagnosed untreated asthma, or has not previously required chronic use of inhaled topical or systemic steroids to control symptoms (cough, wheezing, dyspnea / shortness of breath, or chest pain). In some embodiments, the asthma is chronic corticosteroid-resistant asthma, corticosteroid-refractory asthma, asthma uncontrolled on corticosteroids, or asthma uncontrolled on other chronic asthma controllers. In some embodiments, the autoimmune, inflammatory, fibrotic, neutrophilic, and eosinophilic disorders are pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic), monocytic or lymphocytic disorder is esophagitis, allergic rhinitis, non-allergic rhinitis, rhinosinusitis with polyps, nasal polyps, bronchitis, chronic pneumonia, allergic bronchopulmonary aspergillosis, airway inflammation, allergic rhinitis, bronchiectasis and / or chronic bronchitis.

[0240] In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic and eosinophilic) disorder, monocytic disorder, and lymphocytic disorder is arthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is osteoarthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, early arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, enteropathic arthritis, reactive arthritis, psoriatic arthritis, and / or arthritis as a result of injury.

[0241] In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic and eosinophilic) disorder, monocytic disorder, and lymphocytic disorder are gastrointestinal inflammatory conditions. In some embodiments, the gastrointestinal inflammatory condition is IBD (inflammatory bowel disease), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused by environmental insults (e.g., caused by or associated with therapeutic regimens such as chemotherapy, radiation therapy), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, gastroenteritis, infectious gastritis or enterocolitis (e.g., chronic active gastritis due to Helicobacter pylori infection), and other forms of gastrointestinal inflammation caused by infectious agents, or indeterminate colitis.

[0242] In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic), monocytic disorder, or lymphocytic disorder, or disorder associated with an increase in the number or distribution of normal or abnormal tissue resident cells (such as, for example, mast cells, macrophages, or lymphocytes) or stromal cells (e.g., fibroblasts, myofibroblasts, smooth muscle cells, epithelium, or endothelium), is lupus or systemic lupus erythematosus (SLE), or one or more organ specific manifestations of lupus (e.g., lupus nephritis (LN), which affects the kidneys, or extrarenal lupus (ERL), which affects the blood and / or lymphatic organs (lymph nodes, spleen, thymus, and associated lymphatics), and / or joints and / or other organs, but not necessarily the kidneys).In some embodiments, the autoimmune disorder, inflammatory disorder, or fibrotic disorder is caused by sepsis and / or trauma, HIV infection, or idiopathic (cause unknown), such as ANCA-associated leukoplakia (AAV), granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis), Behcet's disease, cardiovascular disease, eosinophilic bronchitis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), ankylosing spondylitis, dermatomyositis, scleroderma, such as systemic sclerosis, also known as systemic sclerosis, vasculitis (e.g., giant cell arteritis (GCA), also known as temporal arteritis, cranial arteritis, or Horton's disease), myositis, polymyositis, dermatomyositis, polyarteritis nodosa, arteritis, polymyalgia rheumatica, sarcoidosis, primary biliary tract infection, sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus, e.g. pemphigus vulgaris, atherosclerosis, lupus, Still's disease, myasthenia gravis, celiac disease, relapsing remitting (RRMS) or primary progressive (PPMS) or secondary progressive (SPMS) multiple sclerosis (MS), Guillain-Barré disease, diabetes mellitus type 1 (T1DM) or insulin-dependent (IDDM) or early-onset DM type, thyroiditis (e.g. Graves' disease), celiac disease, Churg-Strauss syndrome, myalgia syndrome, hypereosinophilic syndrome, edematous reactions including recurrent angioedema, helminth infections, onchocercal dermatitis dermatitis), eosinophilic esophagitis, eosinophilic enteritis, eosinophilic colitis, obstructive sleep apnoea, endocardial fibrosis, Addison's disease, Raynaud's disease or phenomenon, autoimmune hepatitis, graft versus host disease (GVHD) or organ transplant rejection.

[0243] In a further aspect, the invention provides pharmaceutical formulations comprising any of the bispecific anti-CCL2 antibodies provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the bispecific anti-CCL2 antibodies provided herein and a pharma- ceutically acceptable carrier.

[0244] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, for example, intravenous or subcutaneous, depending in part on whether administration is temporary or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0245] The antibodies of the invention will be formulated, administered, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. Effective amounts of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages as those described herein, or about 1-99% of the dosages described herein, or at any dosages and by any route of administration that are empirically / clinically determined to be appropriate.

[0246] For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) of the antibody can be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment will generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20, or, for example, about 6 doses of the antibody). An initial larger loading dose may be administered, followed by one or more smaller doses. An exemplary dosing regimen includes administering an initial loading dose of about 4 mg / kg, followed by weekly maintenance doses of the antibody of about 2 mg / kg. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0247] II. Manufactured products In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective to treat, prevent, and / or diagnose a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture includes (a) a first container containing a composition, the composition comprising an antibody of the invention, and (b) a second container containing a composition, the composition further comprising a cytotoxic or other therapeutic agent. The article of manufacture, in this embodiment of the invention, may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0248] Specific embodiments of the present invention are listed below. 1. A bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, The bispecific antibody comprises: a) a first polypeptide chain comprising (from N-terminus to C-terminus): VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, VH1 is the first heavy chain variable domain and VL1 is the first variable light chain domain (which together form (associate together) the first antigen-binding site); CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light chain domain, a first polypeptide chain; b) a second polypeptide chain comprising (from N-terminus to C-terminus): VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain, which together form (associate together to form) a second antigen-binding site; CH1 is constant heavy chain domain 1; L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 5 to 10 amino acids); hinge is a heavy chain hinge region; CH2 is constant heavy chain domain 2; CH3 is constant heavy chain domain 3; L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one embodiment, a length of 10 to 15 amino acids); CL is the constant light domain, the second polypeptide chain; Including, A) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or B) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or C) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94; or D) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94; or E) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or F) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94; or G) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or H) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 73; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or I) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or J) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or K) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or L) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or M) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94; or N) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or O) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; or P) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93; Bispecific antibodies.

[0249] 2.i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91; 2. The bispecific antibody of embodiment 1, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93.

[0250] 3.i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71; the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90; 2. The bispecific antibody of embodiment 1, wherein the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94.

[0251] 4. L1 is a polypeptide linker having a length of 9 to 11 amino acids, 4. The bispecific antibody according to any one of embodiments 1 to 3, wherein L2 is a polypeptide linker having a length of 9 to 11 amino acids.

[0252] 5. The bispecific antibody of embodiment 4, wherein L1 and L2 are polypeptide linkers selected from the group of GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185), GGSGGSGGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), and GGGGSGGSGG (SEQ ID NO: 191).

[0253] 6. L1 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188); 5. The bispecific antibody of embodiment 4, wherein L2 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188).

[0254] 7. The bispecific antibody according to any one of embodiments 1 to 6, wherein said constant heavy domains CH1, hinge, CH2 and CH3 are of the human IgG isotype, preferably of the human IgG1 isotype.

[0255] 8. The bispecific antibody, i) Blocking the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC 50 =0.5 nM), and / or ii) inhibited CCL2-mediated chemotaxis of myeloid cells in vitro (IC 50 = 1.5 nM), and / or iii) A bispecific antibody according to any one of embodiments 1 to 7, which is cross-reactive with cynomolgus monkey (cyno) and human CCL2.

[0256] 9. The bispecific antibody according to any one of embodiments 1 to 8, wherein said bispecific antibody is not cross-reactive to other CCL homologues (shows 100-fold lower binding to other CCL homologues (selected from the group of CCL8, CCL7 and CCL13)) compared to binding to CCL2.

[0257] 10. The bispecific antibody according to any one of embodiments 1 to 9, which binds to a first and a second epitope on human CCL2 in an ion-dependent manner.

[0258] 11. The bispecific antibody of any one of embodiments 1 to 9, wherein the bispecific antibody binds to human CCL2 in a pH-dependent manner, and both the first antigen-binding site and the second antigen-binding site bind to CCL2 with higher affinity at neutral pH than at acidic pH.

[0259] 12. The bispecific antibody of any one of embodiments 1 to 10, wherein said bispecific antibody binds to human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

[0260] 13. The constant heavy domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0261] 14. The constant heavy domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L235W, G236N, H268D, Q295L, K326T and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) N434A (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0262] 15. The constant heavy chain domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) N434A (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0263] 16. The constant heavy chain domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and / or P343R (suitable for increasing pI to enhance antigen uptake), and / or ii) L234Y, P238D, T250V, V264I, T307V and / or A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and / or iii) M428L, N434A and / or Y436T (suitable for increasing affinity for FcRn for longer plasma half-life), and / or iv) Q438R and / or S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0264] 17. The constant heavy chain domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0265] 18. The constant heavy chain domains CH1, hinge, CH2 and CH3 are of human IgG1 isotype and contain the following mutations (Kabat EU numbering): i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake), and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgRs), and iii) N434A (suitable for increasing affinity to FcRn for longer plasma half-life), and iv) Q438R and S440E (suitable for inhibiting rheumatoid factor binding) The bispecific antibody according to claim 1 , comprising one or more of the following:

[0266] 19. The constant heavy domain CH3 has the following mutation (Kabat EU numbering): i) S354C and T366W in one of the heavy chain constant domains CH3; and ii) Y349C, T366S, L368A, Y407V in the other heavy chain constant domain CH3 The bispecific antibody according to any one of claims 13 to 18, comprising:

[0267] 20. The constant heavy chain domain CH3 has the following mutation (Kabat EU numbering): K447G The bispecific antibody according to any one of claims 13 to 19, comprising:

[0268] 21. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 175 and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 176.

[0269] 22. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 175 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 176.

[0270] 23. The bispecific antibody according to any one of embodiments 1 to 7, embodiments 1 to 2, and embodiments 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 177, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 178.

[0271] 24. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 177 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 178.

[0272] 25. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 179 and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 180.

[0273] 26. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 179 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 180.

[0274] 27. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 179 and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 180.

[0275] 28. A bispecific antibody according to any one of embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 181 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 182.

[0276] 29. An isolated nucleic acid encoding an antibody according to any one of embodiments 1 to 28.

[0277] 30. A host cell comprising the nucleic acid of embodiment 29.

[0278] 31. A method for producing an antibody, comprising culturing a host cell according to embodiment 30, such that the antibody is produced.

[0279] 32. The method of embodiment 32, further comprising recovering the antibody from the host cell.

[0280] 33. A pharmaceutical formulation comprising a bispecific antibody according to any one of embodiments 1 to 28 and a pharma- ceutically acceptable carrier.

[0281] 34. A bispecific antibody according to any one of embodiments 1 to 28, for use as a medicament.

[0282] 35. A bispecific antibody according to any one of embodiments 1 to 28 for use in the treatment of cancer.

[0283] 36. A bispecific antibody according to any one of embodiments 1 to 28 for use in the treatment of inflammatory and autoimmune diseases.

[0284] 37. Use of a bispecific antibody according to any one of embodiments 1 to 28 in the manufacture of said medicament.

[0285] 38. The use according to embodiment 37, wherein the medicament is for the treatment of cancer.

[0286] 39. The use according to embodiment 37, wherein the medicament is for the treatment of an inflammatory disease or an autoimmune disease.

[0287] 40. A method of treating an individual having cancer, comprising administering to said individual an effective amount of a bispecific antibody according to any one of embodiments 1 to 28.

[0288] 41. A method of treating an individual having an inflammatory or autoimmune disease, comprising administering to said individual an effective amount of a bispecific antibody according to any one of embodiments 1 to 28.

[0289] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0290] Explanation of amino acid sequence Anti-CCL2 antigen-binding moieties (variable and hypervariable regions (CDRs)) that bind to different epitopes: [Table 1] JPEG2024523034000003.jpg255169 CDR modified anti-CCL2 antigen binding portion (variable and hypervariable regions (CDRs)): Modification CNTO888 [Table 2] JPEG2024523034000005.jpg152169 Engineered humanized 11K2 [Table 3] JPEG2024523034000007.jpg255169JPEG2024523034000008.jpg100169

[0291] Exemplary constant light chain regions: SEQ ID NO: 95 Exemplary human kappa light chain constant region SEQ ID NO: 96 Exemplary human lambda light chain constant region

[0292] Exemplary constant heavy chain regions: SEQ ID NO: 97 Exemplary human heavy chain constant region from IgG1 SEQ ID NO: 98 Exemplary human heavy chain constant region from IgG1 with mutations L234A, L235A and P329G (Fc gamma receptor silencing) SEQ ID NO: 99 Exemplary human heavy chain constant region from IgG1 (SG1-IgG1 allotype) SEQ ID NO: 100 Exemplary human heavy chain constant region from IgG1 with mutations (SG105-IgG1 allotype-Fc gamma receptor silencing) SEQ ID NO: 101 SG1095 exemplary human heavy chain constant region from IgG1 containing mutations (Kabat EU numbering): - L235W / G236N / H268D / Q295L / A330K / K326T (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgR); - Q311R / P343R (suitable for increasing the isoelectric point (pI) for enhanced antigen uptake); - N434A (suitable for increasing affinity to FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibiting rheumatoid factor binding) SEQ ID NO: 102 SG1099-Exemplary human heavy chain constant region from IgG1 containing mutations (Kabat EU numbering): Q311R / P343R (suitable for increasing pI to enhance antigen uptake) SEQ ID NO: 103 Exemplary human heavy chain constant region from IgG1 containing SG1100-mutations (Kabat EU numbering): - Q311R / P343R (suitable for increasing pI to enhance antigen uptake); - N434A (suitable for increasing affinity to FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibition of rheumatoid factor binding) CNTO888 / / 11K2-WT IgG1 (Exemplary Bispecific CNTO888 / / 11K2-WT IgG1Crossmab) SEQ ID NO: 104 Heavy chain 1-CNTO888 / / 11K2-WT IgG1 SEQ ID NO: 105 Heavy chain 2-CNTO888 / / 11K2-WT IgG1 SEQ ID NO: 106 Light chain 1-CNTO888 / / 11K2-WT IgG1 SEQ ID NO: 107 Light chain 2-CNTO888 / / 11K2-WT IgG1 CKLO2-IgG1 (Exemplary Bispecific CKLO2IgG1 Crossmab) SEQ ID NO: 108 Heavy Chain 1-CKLO2 IgG1 SEQ ID NO: 109 Heavy Chain 2-CKLO2 IgG1 SEQ ID NO: 110 Light chain 1-CKLO2 IgG1 SEQ ID NO: 111 Light chain 2-CKLO2 IgG1 CKLO2-SG1095 (=P1AD8325) (An exemplary bispecific CLOK2 crossmab containing the SG1095 Fc mutation) SEQ ID NO: 112 Heavy chain 1-CKLO2-SG1095 SEQ ID NO: 113 Heavy chain 2-CKLO2-SG1095 SEQ ID NO: 114 Light chain 1-CKLO2-SG1095 SEQ ID NO: 115 Light chain 2-CKLO2-SG1095 CKLO2-SG1099 (An exemplary bispecific CKLO2 crossmab containing the SG1099 Fc mutation) SEQ ID NO: 116 Heavy chain 1-CKLO2-SG1099 SEQ ID NO: 117 Heavy chain 2-CKLO2-SG1099 SEQ ID NO: 118 Light chain 1-CKLO2-SG1099 SEQ ID NO: 119 Light chain 2-CKLO2-SG1099 CKLO2-SG1100 (An exemplary bispecific CKLO2 crossmab containing the SG1100 Fc mutation) SEQ ID NO: 120 Heavy chain 1-CKLO2-SG1100 SEQ ID NO: 121 Heavy chain 2-CKLO2-SG1100 SEQ ID NO: 122 Light chain 1-CKLO2-SG1100 SEQ ID NO: 123 Light chain 2-CKLO2-SG1100 CKLO3-SG1095 (An exemplary bispecific CLOK3 crossmab containing the SG1095 Fc mutation) SEQ ID NO: 124 Heavy chain 1-CKLO3-SG1095 SEQ ID NO: 125 Heavy chain 2-CKLO3-SG1095 SEQ ID NO: 126 Light chain 1-CKLO3-SG1095 SEQ ID NO: 127 Light chain 2-CKLO3-SG1095 CKLO3-SG1099 (An exemplary bispecific CKLO3 crossmab containing the SG1099 Fc mutation) SEQ ID NO: 128 Heavy chain 1-CKLO3-SG1099 SEQ ID NO: 129 Heavy chain 2-CKLO3-SG1099 SEQ ID NO: 130 Light chain 1-CKLO3-SG1099 SEQ ID NO: 131 Light chain 2-CKLO3-SG1099 CKLO3-SG1100 (An exemplary bispecific CKLO3 crossmab containing the SG1100 Fc mutation SEQ ID NO: 132 Heavy chain 1-CKLO3-SG1100 SEQ ID NO: 133 Heavy chain 2-CKLO3-SG1100 SEQ ID NO: 134 Light chain 1-CKLO3-SG1100 SEQ ID NO: 135 Light chain 2-CKLO3-SG1100

[0293] Further anti-CCL2 antigen-binding moieties: SEQ ID NO: 136 Heavy chain variable domain VH 2F2 SEQ ID NO: 137 Light chain variable domain VL 2F2 SEQ ID NO: 138 Heavy chain variable domain VH Mouse 11K2 (=11K2m) SEQ ID NO: 139 Light chain variable domain VL mouse 11K2 (=11K2m) SEQ ID NO: 140 Heavy chain variable domain VH 1H11 SEQ ID NO: 141 Light chain variable domain VL 1H11

[0294] Exemplary CCL2 and homologs (without signal peptide): SEQ ID NO: 142 Exemplary human CCL2 (MCP1)-wild type (wt) SEQ ID NO: 143 Exemplary human CCL2(MCP1)-P8A variant SEQ ID NO: 144 Exemplary human CCL2(MCP1)-T10C variant SEQ ID NO: 145 Exemplary human CCL8 (MCP2)-wild type (wt) SEQ ID NO: 146 Exemplary human CCL8(MCP2)-P8A variant SEQ ID NO: 147 Exemplary human CCL7 (MCP3)-wild type (wt) SEQ ID NO: 148 Exemplary human CCL13 (MCP4)-wild type (wt) SEQ ID NO: 149 Exemplary cynomolgus CCL2-wild type (wt) SEQ ID NO: 150 Exemplary mouse CCL2-wild type (wt)

[0295] Further exemplary constant heavy chain regions: SEQ ID NO: 151 Exemplary human heavy chain constant region from IgG1 containing GG01-mutations (Kabat EU numbering): - L234Y / P238D / T250V / V264I / T307P / A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgR); - Q311R / P343R (suitable for increasing the isoelectric point (pI) for enhanced antigen uptake); - N434A (suitable for increasing affinity to FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibition of rheumatoid factor binding) SEQ ID NO: 152 Exemplary human heavy chain constant region from IgG1 containing GG02-mutations (Kabat EU numbering): - L234Y / P238D / T250V / V264I / T307P / A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgR); - Q311R / P343R (suitable for increasing the isoelectric point (pI) for enhanced antigen uptake); - M428L / N434A / Y436T (suitable for increasing affinity for FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibition of rheumatoid factor binding) SEQ ID NO: 153 Exemplary human heavy chain constant region from IgG1 (including IgG1 allotype sequences) containing GG03-mutations (Kabat EU numbering): - L234Y / P238D / T250V / V264I / T307P / A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgR); - Q311R / P343R (suitable for increasing the isoelectric point (pI) for enhanced antigen uptake); - N434A (suitable for increasing affinity to FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibition of rheumatoid factor binding) SEQ ID NO: 154 Exemplary human heavy chain constant region from IgG1 (including IgG1 allotype sequences) containing GG04-mutations (Kabat EU numbering): - L234Y / P238D / T250V / V264I / T307P / A330K (suitable for increased affinity for human FcgRIIb and decreased affinity for other human FcgR); - Q311R / P343R (suitable for increasing the isoelectric point (pI) for enhanced antigen uptake); - M428L / N434A / Y436T (suitable for increasing affinity for FcRn for a longer plasma half-life of the antibody); and - Q438R / S440E (suitable for inhibition of rheumatoid factor binding) CKLO2-GG01 (=P1AF8137) Exemplary bispecific CLOK2 crossmab (containing GG01 Fc mutation) SEQ ID NO: 155 Heavy Chain 1-CKLO2-GG01 SEQ ID NO: 156 Heavy chain 2-CKLO2-GG01 SEQ ID NO: 157 Light chain 1-CKLO2-GG01 SEQ ID NO: 158 Light chain 2-CKLO2-GG01 CKLO2-GG02 (=P1AF8139) (Exemplary bispecific CLOK2 crossmab (containing GG02 Fc mutations)) SEQ ID NO: 159 Heavy Chain 1-CKLO2 GG02 SEQ ID NO: 160 Heavy chain 2-CKLO2 GG02 SEQ ID NO: 161 Light chain 1-CKLO2 GG02 SEQ ID NO: 162 Light chain 2-CKLO2 GG02 CKLO2-GG03 (Exemplary Bispecific CLOK2 Crossmab (Containing GG03 Fc Mutation)) SEQ ID NO: 163 Heavy chain 1-CKLO2-GG03 SEQ ID NO: 164 Heavy chain 2-CKLO2-GG03 SEQ ID NO: 165 Light chain 1-CKLO2-GG03 SEQ ID NO: 166 Light chain 2-CKLO2-GG03 CKLO2-GG04 (An exemplary bispecific CKLO2 crossmab containing the GG04 Fc mutation) SEQ ID NO: 167 Heavy chain 1-CKLO2-GG04 SEQ ID NO: 168 Heavy chain 2-CKLO2-GG04 SEQ ID NO: 169 Light chain 1-CKLO2-GG04 SEQ ID NO: 170 Light chain 2-CKLO2-GG04 CKLO2-GG03 / GG04 (=P1AF8140) (an exemplary bispecific CKLO2 crossmab containing the GG03 Fc mutation in the knob chain and the GG04 Fc mutation in the hole chain) SEQ ID NO: 171 Heavy chain 1-CKLO2-GG03 / GG04 SEQ ID NO: 172 Heavy chain 2-CKLO2-GG03 / GG04 SEQ ID NO: 173 Light chain 1-CKLO2-GG03 / GG04 SEQ ID NO: 174 Light chain 2-CKLO2-GG03 / GG04 P1AF8142 (CKLO2-CB-SG1095) - (An exemplary bispecific CKLO2 contour body (CB) containing only two polypeptide chains containing the SG1095 Fc mutation) SEQ ID NO: 175 Polypeptide chain 1-CKLO2-CB-SG1095 SEQ ID NO: 176 Polypeptide chain 2-CKLO2-CB-SG1095 P1AF8143 (CKLO2-CB-GG02) - (An exemplary bispecific CKLO2 contour body (CB) contains only two polypeptide chains containing the GG02 Fc mutation) SEQ ID NO: 177 Polypeptide chain 1-CKLO2-CB-GG02 SEQ ID NO: 178 Polypeptide chain 2-CKLO2-CB-GG02 P1AG5853 (CKLO2-CB-GG02-K447G) - (An exemplary bispecific CKLO2 contour body (CB) contains only two polypeptide chains containing the GG02 Fc mutation and the mutation K447G) SEQ ID NO: 179 Polypeptide chain 1-CKLO2-CB-GG02-KG SEQ ID NO: 180 Polypeptide chain 2-CKLO2-CB-GG02-KG P1AG8317 (CKLO2-CB-SG1095-K447G) - (An exemplary bispecific CKLO2 contour body (CB) containing only two polypeptide chains containing the SG1095 Fc mutation and the mutation K447G) SEQ ID NO: 181 Polypeptide chain 1-CKLO2-CB-SG1095-KG SEQ ID NO: 182 Polypeptide chain 2-CKLO2-CB-SG1095-KG An exemplary glycine-serine polypeptide linker having a length of 10 amino acids: SEQ ID NO: 183 Polypeptide linker GSGGSGGSGG SEQ ID NO: 184 Polypeptide linker GSGGGSGGGG SEQ ID NO: 185 Polypeptide linker GSGGGGSGGG SEQ ID NO: 186 Polypeptide linker GGSGGSGGGG SEQ ID NO: 187 Polypeptide linker GGSGGGSGGG SEQ ID NO: 188 Polypeptide linker GGSGGGGSGG SEQ ID NO: 189 Polypeptide linker GGGSGGSGGG SEQ ID NO: 190 Polypeptide linker GGGSGGGSGG SEQ ID NO: 191 Polypeptide linker GGGGSGGSGG Nomenclature of the monospecific unmodified anti-CCL2 antibodies / antigen-binding moieties used for the anti-CCL2 bispecific antibodies described herein [Table 4]

[0296] Nomenclature of bispecific anti-CCL2 with unmodified VH / VL as a crossmab (see WO 2016 / 016299) with either IgG1 or IgG1 containing the mutations L234A, L235A and P329G (PGLALA) [Table 5]

[0297] Crossmab naming of bispecific antibodies with modified VH / VL (see WO 2016 / 016299). Depending on the heavy chain constant domain used (e.g. IgG1 wild type, PGLALA, SG1095, SG1099, SG1100, GG01, GG02, GG03, GG04, GG02-KG), the following suffix is ​​added: IgG1 wild type, PGLALA, SG1095, SG1099, SG1100, GG01, GG02, GG03, GG04, GG02-KG). [Table 6] TIFF2024523034000012.tif32169 EXAMPLES

[0298] Working Example Example A-1 Monospecific anti-CCL2 antibodies Generation of monospecific anti-CCL2 antibodies and CCL2 antigen

[0299] Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0300] Gene and Oligonucleotide Synthesis The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. The respective oligonucleotides were prepared by Metabion GmbH (Planeg-Martinsried, Germany).

[0301] Description of basic / standard mammalian expression plasmids For expression of the desired genes / proteins (e.g., full-length antibody heavy chain, full-length antibody light chain and CCL-2 molecule), transcription units are used that contain the following functional elements: - the immediate-early enhancer and promoter of human cytomegalovirus (P-CMV) including intron A, - a human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - the genes / proteins to be expressed (e.g. full length antibody heavy and light chains and CCL-2 molecules), and - the bovine growth hormone polyadenylation sequence (BGH pA).

[0302] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains: an origin of replication from the vector pUC18 that allows replication of this plasmid in E. coli, and The beta-lactamase gene that confers ampicillin resistance to Escherichia coli Includes.

[0303] Construction of expression plasmids for recombinant monoclonal antibodies and CCL-2 molecules The expression plasmids for the transient expression of the monoclonal antibodies and the CCL-2 antigen contained, in addition to the respective expression cassettes, the origin of replication from the vector pUC18 allowing replication of this plasmid in E. coli and the beta-lactamase gene conferring ampicillin resistance in E. coli.

[0304] The transcription units for each of the immunoglobulin HC and LC and CCL-2 molecules contained the following functional elements: - the immediate-early enhancer and promoter of human cytomegalovirus (P-CMV) including intron A, - a human heavy chain immunoglobulin 5' untranslated region (5'UTR), - a mouse immunoglobulin heavy chain signal sequence, and - the bovine growth hormone polyadenylation sequence (BGH pA).

[0305] Based on the VH and VL, each of the antibodies 1A4, 1A5, 1G9, 2F6, CNTO888, murine and humanized 11K2, ABN912 was generated as IgG1 wild type and as IgG1 PGLALA / effector silent Fc with a kappa light chain. Transient expression and purification Recombinant production was performed by transient transfection of HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For production of monoclonal antibodies, cells were co-transfected with plasmids containing the respective immunoglobulin heavy and light chains. For transfection "293-fectin", a transfection reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were harvested 3-7 days after transfection. Supernatants were stored at low temperature (e.g., -80°C).

[0306] For example, general information regarding recombinant expression of human immunoglobulins in HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

[0307] Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured onto MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate, pH 3.0. Eluted protein fractions were pooled, neutralized with 2 M Tris, pH 9.0, and further purified by size exclusion chromatography using a Superdex 200 26 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Size-exclusion chromatography fractions were analyzed by CE-SDS (Caliper Life Science, USA) and antibody-containing fractions were pooled and stored at -80°C.

[0308] Generation of recombinant CCL2 Wild-type CCL2 can exist as a monomer, but can also form dimers at physiological concentrations. This monomer-dimer equilibrium may be different and must be carefully considered for all described in vitro experiments in which different concentrations may be used. To avoid uncertainty, we created a point-mutated CCL2 variant: the P8A variant of CCL2 carries a mutation in the dimerization interface and is therefore unable to form dimers resulting in a defined pure CCL2 monomer. In contrast, the T10C variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20;135(11):4325-32).

[0309] Each soluble CCL2 protein (wild type, P8A and T10C variant) was purified from cell culture supernatant by cation exchange chromatography using SP-Sepharose HP (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatant was diluted with 10 mM KH2PO4, pH 5.0 to adjust the conductivity to <4 mS / cm. The diluted supernatant was loaded onto SP-Sepharose resin equilibrated with 10 mM KH2PO4, pH 5.0, washed with equilibration buffer and eluted with a gradient to 10 mM KH2PO4, 1 M NaCl, pH 5.0. The eluted protein fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 16 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Size-exclusion fractions were analyzed by SDS-PAGE and analytical high performance size-exclusion chromatography. CCL2-containing fractions were pooled and stored at -80°C.

[0310] Functional characterization (binding) The T200 instrument was fitted with a Biacore Series S Sensor Chip CM5. The system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20). The system was set at 37° C. For each measurement, the sample buffer was the system buffer further supplemented with 1 mg / ml CMD (carboxymethyl dextran, Fluka).

[0311] An antibody capture system was established. 14000 GARFcγ (goat anti-rabbit Fcγ), 111-005-046, Jackson ImmunoResearch) was immobilized at 25 μg / ml in 10 mM sodium acetate buffer pH 5.0 at 25° C. by EDC / NHS coupling as described by the manufacturer. The capture system was regenerated at 20 μl / min by a 15 s injection with HBS buffer (100 mM HEPES pH 7.4, 1.5 M NaCl, 0.05% (w / v) Tween 20), a 1 min injection with 10 mM glycine buffer pH 2.0, followed by two 1 min injections with 10 mM glycine buffer pH 2.25. In another embodiment, mouse monoclonal antibodies were captured on the biosensor by immobilizing 12700 RU polyclonal rabbit anti-mouse (RAM IgG, GE Healthcare) antibodies on the Biacore series CM5 sensor as described above. The sensor was regenerated by a 3 min injection of 10 mM glycine buffer pH 1.7.

[0312] Antibody clone supernatants were diluted 1:2 in system buffer and captured at 5 μl / min for 1 min. After antibody capture, the system was washed with 2.5x concentrated system buffer at 80 μl / min for 30 s followed by 2 min baseline stabilization. Analyte kinetics were performed at 30 μl / min. Human CCL2 and monomeric CCL2 P8A variant CCL2 were used as analytes in solution wt. Analytes were injected at a maximum concentration of 90 nM. Analyte contact time was 3 min and dissociation time was 10 min. Biaevaluation software V.3.0 was used according to the manufacturer GEHC's instructions. Kinetic rates were estimated apparently applying a 1:1 binding model with RMAX localization. Binding of antibodies to wild-type (wt) human CCL2 and human CCL2 P8A variant (monomer) [Table 7]

[0313] Summary of pH-dependent CCL2 binding kinetics obtained from SPR analysis I

[0314] The T200 instrument was fitted with a Biacore Series S Sensor Chip CM5. The system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20). In other embodiments, the pH of the system buffer was set to pH 8.3, pH 7.9, pH 7.4, pH 7.1, pH 6.7, pH 6.3, pH 5.9, pH 5.5. The system was set at 25° C. For each measurement, the sample buffer was the system buffer further supplemented with 1 mg / ml CMD (carboxymethyl dextran, Fluka).

[0315] An antibody capture system was established. 13000MAb <h-fc-pan>M-R10Z8E9-IgG (Roche) was immobilized at 18 μg / ml in 10 mM sodium acetate buffer pH 5.0 at 25° C. by EDC / NHS coupling as described by the manufacturer. The capture system was regenerated by injection of HBS buffer (100 mM HEPES pH 7.4, 1.5 M NaCl, 0.05% (w / v) Tween 20) at 20 μl / min, followed by 15 s injections of 10 mM NaOH for 1 min and two injections of 10 mM glycine buffer pH 2.5 for 1 min. The captured antibodies were injected at 10 μl / min at 80 nM concentration diluted in the respective system buffer for 30 s. After antibody capture, the system was washed with 2.5x concentrated system buffer at 50 μl / min for 30 s, followed by 2 min of baseline stabilization. A concentration-dependent analyte series was injected in 1:3 dilution steps from 0 nM (buffer control), 0.4 nM, 1.1 nM, 3.3 nM, 30 nM in duplicate injections. Analyte contact time was 3 min and dissociation time was 10 min. Analyte kinetics were performed at 50 μl / min.

[0316] Human antibodies were captured as ligands on the sensor surface: ● Human normal IgG (HN-IgG, Id.:11717570, Roche) as a positive control; ● Anti-human CCL2 mAb (humanized 11k2: CCL2-0002), ● Anti-human CCL2 mAb (AB912, CCL2-0003), and ● Anti-human CCL2 mAb (CNTO888, CCL2-0004); - System buffer as a negative control.

[0317] The Biaevaluation software V.3.0 was used according to the instructions of the manufacturer GEHC. MAX A localized 1:1 binding model was applied to determine the kinetic rates. [Table 8]

[0318] Cross-reactive CCL homologues Since CCL2 (MCP-1) has high homology to CCL7 (MCP-3), CCL8 (MCP-2), and CCL13 (MCP-4), and these CCL chemokines can bind to CCR2, the binding of anti-CCL2 antibodies to these homologs was evaluated. The results are shown in Figure 1. Except for CNTO888 (Mol Immunol. 2012 Jun;51(2):227-33), which was described as having selectivity for CCL2, the other antibodies tested bound to either CCL7 or CCL8 (showed cross-reactivity to either CCL7 or CCL8).

[0319] Biacore assay: Binding of anti-CCL2 antibodies to CCL homologs such as CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3) and CCL13 (MCP-4) was assessed at 25°C using a Biacore T200 instrument (GE Healthcare). Mouse anti-human IgG (Fc) (GE Healthcare) was immobilized on each flow cell of a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1 mg / ml BSA, 0.05% Tween20, 0.005% NaN3). Antibodies were captured on the anti-Fc sensor surface and then recombinant human CCL homolog proteins were injected at 5 nM and 20 nM over the flow cells. Wild-type CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3) and CCL13 (MCP-4) were commercially available from R&D Systems, while monomeric CCL2 (P8A variant) was an in-house generated antigen. The sensor surface was regenerated with 3 M MgCl2 at each cycle. Binding sensorgrams were processed using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0320] Functional characterization (biological) CCR2 signaling I-Calcium flux assay THP-1 (human acute monocytic leukemia cell line; ATCC TIB-202) cells were cultured in RPMI 1640, 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM β-mercaptoethanol (supplied by Thermo Fisher Scientific). On the day of the assay, the cell density was 8.33 × 10 in 25.8 ml assay medium (RPMI 1640 without FBS). 5 The cells were adjusted to 1000 cells / ml. FLIPR® Calcium Assay Kits (FLIPR Calcium 4 Assay Kit, Catalog No. R8142,

[0321] The dye loading solution was prepared by mixing two vials of component A with 20 ml of component B (HBSS buffer with 20 mM HEPES, pH 7.4) according to the manufacturer's instructions, Molecular Devices. 516 μl of 1 M Hepes (final assay concentration: 10 mM) is added, followed by 516 μl of 250 mM probenecid (final assay concentration: 2.5 mM). For the stock solution, 65.4 mg of probenecid (Sigma P8761) is dissolved in 465 μl of 1 N NaOH and 465 μl of 1×HBSS (Thermo Fisher Scientific) is added. 25.8 ml of loading buffer is added to, for example, 4 microtiter plates (52.6 ml volume is required; 10 6 The cells were mixed with 25.8 ml of assay medium containing enough cells to culture 100 µl of THP-1 / ml. 120 µl of the cell suspension in loading buffer was transferred to each well of a black F-bottom 96-well cell culture plate. The plate was incubated at room temperature for 3-4 hours.

[0322] Meanwhile, antibody and ligand solutions were prepared. Eight concentrations of each antibody were tested, ranging from 30 μg / ml to 0.025 μg / ml (no serial dilutions, final concentration in wells). Each concentration was tested in two plates. All dilutions were prepared as 10x concentrated solutions in assay medium. As a reference antibody, human CCL2 / JE / MCP-1 antibody (R&D Systems catalogue no. MAB279) was used. Ligand CCL2 (R&D Systems catalogue no. 279-MC-10) was prepared by dissolving 50 μg of CCL2 lyophilisate in 500 μl of RPMI1640 (100 μg / ml) and transferring 400 μl to 10 ml of assay medium (4 μg / ml stock solution). Ionomycin (Sigma Cat. No. I-0634) was used as a stimulation control (1 mg ionomycin, 1 mM dissolved in 1340 μl DMSO (Sigma Cat. No. D-8779)). 10 μl of the 1 mM stock solution was diluted in 1990 μl assay medium (5 μM, final assay concentration 500 nM). 100 μl was pipetted into the corresponding control wells of the polypropylene MTP.

[0323] Antibody dilutions and CCL2 were pre-incubated in two V-shaped polypropylene 96-well plates. 50 μl of 4 μg / ml stock solution CCL2 (final 400 ng / ml CCL2) and 50 μl of 10-fold concentrated antibody dilutions were pipetted into the wells. Plates were incubated at room temperature for 30-60 minutes.

[0324] After incubation, the cell plate and compound plate were transferred directly to the reading position of the FlexStation®3 (Molecular Devices) and the calcium assay was performed as described in the system manual (Excitation 485 nm, Emission 525 nm). Readings were taken at intervals of a few seconds.

[0325] result: [Table 9]

[0326] Efficacy of anti-CCL2 antibodies to inhibit CCL2-induced internalization of the CCR2 receptor expressed on monocytes. To prevent ligand-induced CCR2 internalization on bone marrow cells, we set up an in vitro assay and characterized anti-CCL2 antibodies. Monocytes were isolated by magnetic separation from peripheral blood of healthy donors using a commercial kit (Stemcell, Cat. No. 15068). For blocking of FcγR, monocytes were pre-incubated with normal human IgG (Privigen, CSL Behring) at a final concentration of 500 μg / ml in FACS buffer (PBS + 0.2% BSA) for 50 min on ice. Cells were then centrifuged for 10 min (300xg, 4°C), washed once more with FACS buffer and stored on ice. Anti-CCL2 antibody dilutions (50 μl each) were prepared in 96U bottom wells (BD) (parallel approach at 4°C and 37°C). Monocytes were split, resuspended in culture medium (RPMI1640; 10% FCS; 2 mM L-glutamine) and incubated at 4°C and 37°C, respectively, until further use. Recombinant CCL2 (50 μl; at a final concentration of 100 ng / ml) was added to the prepared antibody dilutions at both 4°C and 37°C. 100 μl of monocyte suspension (2 × 10 5 Cells / well) were added to the CCL2 / anti-CCL2 mixture in a total volume of 200 μl and the cells were incubated at 4° C. and 37° C. for 1 h 30 min before centrifugation at 300×g at 4° C. From this point on, all steps were performed with pre-chilled buffer: cells were washed with 250 μl FACS buffer and cells were further stained for the CCR2 receptor (using a commercially available CCR2-APC conjugate and the appropriate isotype ctrl-APC according to standard FACS protocols: aliquots were added to CD192(CCR2)APC (BioLegend, #357208, clone K036C2 / mIg G2aκ) and the appropriate isotype ctrl antibody: 20 μl / 10 6 Cells mIgG2a k APC BD Biosciences, #400222, clone MOPC-173, 5μl / 10 6 cells were stained).

[0327] Receptor expression was then analyzed by FACS Canto II and CCR2 internalization was calculated as follows: • No internalization: cells were analyzed upon incubation in the absence of ligand (rec.CCL2). • 100% internalization: maximal reduction in CCR2 expression levels on cells previously incubated with rec.CCL2. [Table 10]

[0328] Inhibition of CCL2-mediated chemotaxis towards human THP-1 cells CCR2 towards a CCL2 gradient + THP1 cell migration was tested as follows: Monocytic THP1 cells (ATCC® TIB-202™) were cultured in RPM1 1640 medium (PAN, catalog number P04-17500) supplemented with FCS and L-glutamine. Cells were usually passaged 2-3 times before use in migration assays and then starved overnight in medium with reduced FSC content (1.5% instead of 10% FCS). Cells were counted and incubated with 10 μg / ml normal human IgG (Invitrogen, catalog number 12000; blocks FcgR) for 15 min at room temperature.

[0329] Meanwhile, anti-CCL2 antibodies (and / or controls) were added to the lower chamber of an HTS Transwell 96-well plate system (Corning, Cat. No. 3386; 3 μm pore size) containing serum-free medium with 25 ng / ml rhCCL-2 (R&D Systems, Cat. No. #279-MC). The insert plate was then attached to the lower chamber-plate and 75 μl (1.5 × 10 5 The above cell suspension (containing IgG block) was added to each insert with and without 5 μg / ml antibody / isotype. The plates were covered and incubated overnight at 37° C. in a CO2 incubator (5% CO2).

[0330] The insert plate was removed and Cell-titer-glo substrate (Promega, Cat. No. G758) was added to each well of the lower chamber plate to measure the viability of the migrated cells. After 1 hour of incubation on a shaker at 300 rpm (cover plate was sealed), 200 μl from each well was transferred to a Microfluor black 96-well plate (VWR, Cat. No. #735-0527) and luminescence was measured (luminescence reader, e.g. Bio-Tek, Tecan). The fold change was calculated as the ratio between the number of migrated cells (Cell Titer Glo, RLU) with IgG control antibody and anti-CCL2 antibody. Table 2 below shows the results of 5-10 replicates per condition. [Table 11]

[0331] Assessment of human CCL2 immune complex clearance by monospecific (monoparatopic) anti-CCL2 antibodies in mice To evaluate the ability of monoparatopic antibodies to form immune complexes with wild-type human CCL2, preformed immune complexes consisting of anti-CCL2 monoparatopic antibodies (20 mg / kg) and wild-type human CCL2 (0.1 mg / kg) were injected into human FcRn transgenic mice (B6.Cg-Fcgrt) at a single dose of 10 ml / kg. tm1Dcr Tg(FCGRT)32Dcr / DcrJ, Jackson Laboratory) into the tail vein. Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. Serum was prepared by immediately centrifuging the blood at 14,000 rpm for 10 minutes at 4°C. Serum was stored at -80°C or below until measurement. The monoparatopic antibodies tested are listed in Table 3 below. Antibodies with SG1 Fc have Fc gamma receptor binding similar to wild type, while antibodies with SG105 Fc are Fc gamma receptor binding silent.

[0332] As shown in Figure 2, the effect of immune complex sweeping of each anti-CCL2 monoparatopic antibody on hCCL2 clearance in vivo was evaluated by comparing anti-CCL2 antibody with Fc gamma receptor binding (SG1, = IgG1 wild type with intact Fc gamma receptor binding; solid line) and anti-CCL2 antibody with Fc gamma receptor binding silent (SG105, = IgG1 without Fc gamma receptor binding; dotted line). Each Figure 2a-g shows the serum concentration of hCCL2 over time after injection of preformed immune complexes consisting of hCCL2 and the respective anti-CCL2 antibody (two different Fc moieties: SG1 = IgG1 wild type with intact Fc gamma receptor binding, and SG105 = IgG1 without Fc gamma receptor binding) in FcRn transgenic mice. The antibody profile was analyzed by noncompartmental analysis using Phoenix 64 (Pharsight / Certara). AUCinf was estimated by the linear logarithmic trapezoidal rule extrapolated to infinity. The clearance value is defined as dose / AUCinf. The clearance difference was also expressed as a fold change calculated by dividing the hCCL2 clearance of the antibody with Fc gamma receptor binding (SG1) by the hCCL2 clearance of the antibody with Fc gamma receptor binding silent (SG105) (Table 3 below). The data in Table 3 below show that the clearance of human CCL2 by Fc gamma receptor binding antibodies (SG1 = IgG1 wild type with intact Fc gamma receptor binding) was similar to the clearance by Fc gamma receptor binding silent antibodies (SG105 without Fc gamma receptor binding) for all monoparatopic antibodies tested. This suggests that immune complex-mediated scavenging of CCL2 by the monoparatopic antibodies tested was not efficient. [Table 12]

[0333] Measurement of total human CCL2 concentrations in serum by electrochemiluminescence (ECL) The concentration of total human CCL2 in mouse serum was measured by ECL. 3ug / mL of anti-CCL2 antibodies (F7 (Biolegend) and clone MAB679 (R&D Systems)) were immobilized on a MULTI-ARRAY 96-well plate (Meso Scale Discovery) overnight, followed by incubation in blocking buffer at 30°C for 2 hours. Anti-CCL2 MAB679 was used as the capture antibody for samples containing humanized 11K2, 1A4 and 1A5 antibodies. Anti-CCL2 clone 5D3-F7 was used for samples containing ABN912, CNTO888, 1G9, 2F6H antibodies. Human CCL2 standard curve samples, quality control samples and mouse serum samples were prepared by diluting in dilution buffer and incubating with excess drug for 30 minutes at 37°C. Samples were then added to the anti-CCL2 immobilized plate and allowed to bind for 1 hour at 30°C, followed by washing. SULFO TAG NHS-ester (Meso Scale Discovery)-labeled anti-human Fc (clone: ​​JDC-10, SouthernBiotech) was then added and the plate was incubated for 1 h at 30° C. and then washed. Readout buffer T (×4) (Meso Scale Discovery) was immediately added to the plate and the signal was detected by a SECTOR Imager 2400 (Meso Scale Discovery). Human CCL2 concentrations were calculated based on the standard curve response using the analysis software SOFTmax PRO (Molecular Devices).

[0334] Measurement of serum anti-CCL2 antibody concentrations by enzyme-linked immunosorbent assay (ELISA) The concentration of anti-CCL2 antibodies in mouse serum was measured by ELISA. Anti-human IgG κ chain (Antibody Solutions) was dispensed into a Nunc MaxiSorp plate (Thermofisher) and left to stand overnight at 4°C to prepare an anti-human IgG immobilized plate. A calibration curve and samples were prepared with 1% pooled mouse serum. Then, the samples were dispensed into the anti-human IgG immobilized plate and left to stand at 30°C for 1 hour. Then, goat anti-human IgG (gamma chain specific) with HRP conjugate (Southern Biotech) was added and reacted at 30°C for 1 hour. The color reaction was performed using TMB substrate (Life Technologies) as a substrate. After stopping the reaction with 1N sulfuric acid (Wako), the absorbance at 450 nm was measured with a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

[0335] Assessment of endogenous murine CCL2 immune complex scavenging by monoparatopic antibodies in mice In addition to the above results (suggesting that immune complex-mediated scavenging of CCL 2 by the monoparatopic antibodies tested was not efficient), further evaluation was performed.

[0336] To evaluate the ability of monoparatopic antibodies to form and clear immune complexes with endogenous mouse CCL2, mice were administered mouse cross-reactive 11K2 anti-CCL2 monoparatopic antibodies. Humanized 11K2H2-SG1 (IgG1 wild type = Fc gamma receptor binding) and humanized 11K2-SG105 (Fc gamma receptor binding silent) antibodies were administered intravenously into the tail vein of Balb / c mice at a single dose of 20 mg / kg and a single dose of 10 ml / kg. Blood was collected before administration, 5 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. Serum was prepared by immediately centrifuging the blood at 14,000 rpm for 10 minutes at 4°C. Serum was stored at or below -80°C until assayed.

[0337] FIG. 3 shows the time course of serum total mouse CCL2 concentrations and antibody-time profiles of humanized 11K2-SG1 and 11K2-SG105 in mice.

[0338] As seen in Figure 3, the levels of accumulated mouse CCL2 were not different between 11K2-SG105 (Fc gamma receptor binding silent Fc) and 11K2-SG1 (IgG1 wild type = Fc gamma receptor binding Fc). This indicates that there was little or no Fc gamma receptor mediated clearance of endogenous mouse CCL2 by the injected antibody. Since antigens in immune complexes are cleared more quickly than uncomplexed antigens via multimeric binding of Fc gamma receptors, this suggests that the 11K2 antibody was not able to form immune complexes with endogenous mouse CCL2.

[0339] Measurement of mouse CCL2 concentrations in mouse serum by enzyme-linked immunosorbent assay (ELISA) The concentration of mouse CCL2 in mouse serum was measured by adapting the reagents from a commercially available mouse CCL2 ELISA kit (R&D Systems). The manufacturer's protocol was followed, except for the preparation of standard curve samples. Purified recombinant mouse CCL2 was substituted as a standard instead of the manufacturer's protein. For samples taken after antibody injection, standard curve samples and samples were prepared with 2.5% mouse serum injection antibody spiked at a concentration of 40 micrograms / ml and incubated at 37°C for 30 minutes. Samples were then dispensed onto anti-human CCL2 immobilized plates and incubated at 30°C for 2 hours. Detection by adding mouse MCP-1 conjugate and incubating at 30°C for 2 hours, followed by addition of substrate and stop solution.

[0340] For samples taken prior to antibody injection, the Mouse MCP-1 Ultra-Sensitive Kit (Meso Scale Discovery) was used according to the manufacturer's instructions. No antibody was added to the samples prior to addition to the plate.

[0341] Measurement of serum anti-CCL2 antibody concentrations by enzyme-linked immunosorbent assay (ELISA) The concentration of anti-CCL2 antibody in mouse serum was measured by ELISA. Anti-human IgG κ chain (Antibody Solutions) was dispensed into a Nunc MaxiSorp plate (Thermofisher) and left to stand overnight at 4°C to prepare an anti-human IgG immobilized plate. A calibration curve and samples were prepared with 1% pooled mouse serum. Then, the samples were dispensed into the anti-human IgG immobilized plate and left to stand at room temperature for 1 hour. Then, mouse anti-human IgG HRP (clone JDC-10, Southern Biotech) was added and reacted at room temperature for 30 minutes. A color reaction was performed using ABTS substrate (KPL) as a substrate, and the absorbance at 405 nm was measured with a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

[0342] Conclusions of different mouse PK studies using monospecific (monoparatopic) anti-CCL2 antibodies To summarize the results of the mouse PK studies, none of the monoparatopic antibodies tested demonstrated efficient clearance of CCL2 from the circulation. These data suggest that the monoparatopic antibodies are unable to form immune complexes with CCL2 to efficiently remove it from the circulation.

[0343] In contrast, as described below, a bispecific anti-CCL2 antibody bearing two different antigen-binding moieties / sites (biparatopic anti-CCL2 antibody) was able to efficiently form immune complexes with CCL2 and remove it from the circulation.

[0344] Example B-1 Bispecific (biparatopic) anti-CCL2 antibodies Several bispecific anti-CCL2 antibodies have been generated that have two different antigen-binding moieties (paratopes) that bind to two different specific epitopes on human CCL2.

[0345] Introduction To test whether single binding and cross-linking of antigens has a significant effect on in vivo CCL2 clearance, we used bispecific CrossMab Technology (see, e.g., WO 2009 / 080252, WO 2015 / 150447), WO 2009 / 080253, WO 2009 / 080251, WO 2016 / 016299, Schaefer et al, PNAS, 108(2011)1187-1191, and Klein at al., MAbs 8(2016)1010-20) (bispecific (=biparatopic) CrossMab) to generate bispecific anti-CCL2 antibodies with two different antigen-binding moieties / sites that bind to two different epitopes on CCL2. These molecules were initially characterized for their biochemical and functional properties in vitro, but also served as tools for in vivo CCL2 clearance assessment in mouse co-injection studies. To assess clearance capabilities based on Fc gamma receptor (FcgR) binding-mediated scavenging (e.g., Igawa et al, Immunological Reviews 270(2016)132-151, WO 2012 / 122011, and WO 2016 / 098357, and WO 2013 / 081143), we generated all crossmasbs as wild-type huIgG1 that binds to FcgR, and with modified human IgG1 constant chains that have reduced / abolished binding to FcgR effector-silent molecules (e.g., IgG1 with mutations L234A, L235A, P329G (Kabat EU numbering)).

[0346] Identification of suitable anti-CCL2 antibody pairs - Selection of biparatopic antibody arms by sandwich ELISA. A sandwich ELISA was performed to identify antibody pairs that do not compete for binding to human CCL2. 384-well MAXISORP (NUNC) plates were coated with 1 μg / mL of the seven indicated capture antibodies (arm 1) and blocked with 2% BSA. Biotinylated (NHS-PEO4-biotin, Pierce) WT human CCL2 (20 ng / mL) was incubated with an excess of the same seven antibodies (arm 2) at 1 μg / mL and blocking buffer for 1 h at 37 degrees Celsius. After incubation, the mixture was added to the blocked ELISA plate and incubated at room temperature for 1 h. Detection of plate-bound CCL2 was performed using streptavidin-HRP followed by TMB One Component substrate (Lifetech). Signal development was stopped by 1N HCl acid (Wako). The OD of wells without competing antibodies was set as 100% signal for each capture antibody. The OD of blank wells without added CCL2 was set as 0% signal. Nine antibody pairs that did not show strong competition for CCL2 binding in both orientations were selected as candidates for the generation of bispecific crossmab antibodies. [Table 13]

[0347] Generation and characterization of biparatopic anti-CCL2 antibodies and immune complexes Generation of biparatopic anti-CCL2 antibodies in bispecific CrossMab format recombinant DNA technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0348] Gene and Oligonucleotide Synthesis The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. The respective oligonucleotides were prepared by Metabion GmbH (Planeg-Martinsried, Germany).

[0349] Description of basic / standard mammalian expression plasmids For expression of a desired gene / protein (eg, an antibody heavy chain or an antibody light chain), a transcription unit containing the following functional elements is used: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) containing intron A; ● Human heavy chain immunoglobulin 5' untranslated region (5'UTR), ● Mouse immunoglobulin heavy chain signal sequence, The gene / protein to be expressed (e.g., a full-length antibody heavy chain or an MHC class I molecule), and • Bovine growth hormone polyadenylation sequence (BGH pA). In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid is the origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and • It contains the beta-lactamase gene that confers ampicillin resistance to E. coli.

[0350] 2. Preparation of Expression Plasmids for Recombinant Monoclonal Antibodies The recombinant monoclonal antibody genes encode respective heavy and light immunoglobulin chains.

[0351] The expression plasmids for transiently expressing monoclonal antibody molecules contained, in addition to the immunoglobulin heavy or light chain expression cassette, an origin of replication from the vector pUC18 allowing replication of this plasmid in E. coli and a beta-lactamase gene conferring ampicillin resistance in E. coli.

[0352] Each antibody heavy or light chain transcription unit contained the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) containing intron A; ● Human heavy chain immunoglobulin 5' untranslated region (5'UTR), ● Mouse immunoglobulin heavy chain signal sequence, - the respective antibody heavy and light chain cDNA sequences, and • Bovine growth hormone polyadenylation sequence (BGH pA).

[0353] Transient expression and analytical characterization Recombinant production was performed by transient transfection of HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For production of monoclonal antibodies, cells were co-transfected with plasmids containing the respective immunoglobulin heavy and light chains. For transfection "293-fectin", a transfection reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were harvested 3-7 days after transfection. Supernatants were stored at low temperature (e.g., -80°C).

[0354] General information on recombinant expression of human immunoglobulins, for example in HEK293 cells, is given below: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203. To generate the bispecific antibodies described below, the CrossMab technology described in WO 2016 / 016299 was used, in which VH / VL was exchanged in one antibody arm and the CH1 / CL interface of the other antibody arm was modified by charge modification in combination with knobs-into-holes technology at the CH3 / CH3 interface to promote heterodimerization. Exemplary sequences of all four antibody chains to which this technology was applied are given for CNTO888 / / 11K2-WT IgG1 (see SEQ ID NO: 104 to SEQ ID NO: 107).

[0355] List of bispecific (biparatopic) anti-CCL2 crossmab antibodies generated using wild-type IgG1 (WT IgG1) (wild-type IgG1 means no modifications / mutations affecting Fc receptor binding, but includes heterodimerization techniques like knobs-into-holes). [Table 14]

[0356] List of bispecific (biparatopic) anti-CCL2 crossmab antibodies with IgG1 containing Fc gamma receptor silencing mutations L234A, L235A, P329G (Kabat EU numbering) (IgG1-PGLALA) [Table 15]

[0357] Purification of biparatopic anti-CCL2 antibodies

[0358] The biparatopic anti-CCL2 antibody containing cell culture supernatant was filtered and purified by up to three chromatographic steps. Depending on the purity of the capture step eluate, an ion exchange chromatography step may be performed between the capture step and the final purification step.

[0359] Biparatopic anti-CCL2 antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden), POROS 50 HS (Thermofisher Scientific) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate, pH 3.0. Eluted precursor fractions were pooled and neutralized with 2 M Tris, pH 9.0.

[0360] Ion exchange chromatography was performed as an optional second purification step using a POROS 50 HS (Thermofisher Scientific), equilibrated, washed with 20 mM histidine pH 5.6, loaded with diluted capture step eluate, and gradient chromatography was performed with 20 mM histidine, 0.5 M NaCl at pH 5.6. Ion exchange chromatography fractions were analyzed by CE-SDS LabChip GX II (PerkinElmer) and Crossmab-containing fractions were pooled.

[0361] Size exclusion chromatography on Superdex 200 (GE Healthcare) was used as the second and third purification steps. Size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by CE-SDS LabChip GX II (PerkinElmer) and Crossmab-containing fractions were pooled and stored at -80°C.

[0362] If product quality was met, size exclusion chromatography (Superdex 200 (GE Healthcare)) was replaced by desalting chromatography on a POROS50HS (ThermoFisher Scientific) followed by HiPrep 26 / 10 desalting (GE Healthcare) in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0.

[0363] The protein concentration of the antibody formulations was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence.

[0364] Antibody purity and integrity were analyzed by CE-SDS using a LabChip GX II (PerkinElmer) with a Protein Express Chip and HT Protein Express Reagents Kit. Aggregate content of antibody preparations was determined by high performance SEC using a Biosuite High Resolution SEC, 250 Å, 5 μm analytical size exclusion column (Waters GmbH) with 200 mM K2HPO4 / KH2PO4, 250 mM KCl, pH 7.0 as running buffer. Average purity was between 94-100% as analyzed by CE-SDS, with monomer content >95% (SEC).

[0365] Functional characterization of bispecific (biparatopic) anti-CCL2 antibodies Affinity measurement (binding) Approximately 1200 resonance units (RU) of the capture system (20 μg / ml goat anti-human IgG Fc; order code: 109-005-098; Jackson Immuno Research) were coupled onto a C1 chip (GE Healthcare BR-1005-35) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. Sample and system buffer was PBS-T (10 mM phosphate buffered saline with 0.05% Tween 20) pH 7.4. The flow cell was set at 25° C. and the sample block was set at 12° C. and primed twice with running buffer. The bispecific antibody was captured by injecting a 2 μg / ml solution at a flow rate of 10 μl / min for 60 seconds. Association was measured by injection of various concentrations of human CCL2 (wt) in solution starting with a 1:10 dilution of 30 nM for 150 seconds at a flow rate of 30 μl / min. The dissociation phase was monitored for up to 1200 s and was triggered by switching from the sample solution to running buffer. The surface was regenerated by washing with 0.85% H3PO4 solution at a flow rate of 10 μl / min for 60 s. Bulk refractive index differences were corrected by subtracting the response obtained from a goat anti-human IgG Fc surface. A blank injection was also subtracted (double referenced). A Langmuir 1:1 model was used to calculate the kinetic parameters. [Table 16]

[0366] Formation of innate immune complexes in the presence of wild-type antigen. All protein samples (bispecific anti-CCL2 CrossMab antibodies and antigens) were rebuffered in 1×PBS, pH 7.4 using dialysis and centrifugal ultrafiltration devices.

[0367] A dilution series of CrossMab samples from 2.0 to 0.1 mg / mL was prepared. Similarly, antigen solutions in PBS were prepared at concentrations ranging from 0.012 to 0.23 mg / mL. Concentrations were chosen to allow mixing of equal volumes to achieve a constant molar ratio of 1:1 (antibody:CCL2 complex). The following antigens were used in this study: wild-type CCL2.

[0368] Equal volumes of pre-diluted CrossMab and CCL2 preparations were mixed and incubated for 1 h at 37° C., after which the sample was applied to a Superose 6 (GE Healthcare #2039) column, pre-equilibrated with PBS, and eluted at a flow rate of 0.5 mL / min. A total of 100 μg and the maximum possible volume of 250 μL were applied, and antibody and antigen alone were used as controls.

[0369] SEC-MALLS data were recorded using an OptiLab rEX refractive index detector and a miniDAWN Treos MALLS detector, both from Wyatt Inc. SEC-MALLS signals were processed using Astra V5 software (Wyatt). [Table 17]

[0370] CCR2 reporter assay to study the neutralizing properties of anti-CCL2 antibodies Tango™ CCR2-bla U2OS cells were purchased from Invitrogen (Germany) to test the effect of CCL2 neutralizing antibody constructs. These reporter cells contain human chemokine (CC motif) receptor 2 (CCR2) linked to a TEV protease site and Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parent cell line, which stably expresses a β-arrestin / TEV protease fusion protein and a β-lactamase (bla) reporter gene under the control of a UAS response element. Addition of the natural ligand MCP1=CCL2 provides an indication of reporter gene activity, which can be measured by cleavage of a FRET-compatible substrate.

[0371] Essentially, the assay and cell handling procedures were as described in the supplier's manual. Briefly, CCR2-U2 OS cells were cultured at 2 × 10 in 50 μl of assay medium (Freestyle 293 Expression Medium, Cat. No. 12338-018, Invitrogen). 4 Cells were seeded at a density of 10 ...

[0372] The next day, CCF4 substrate (Cat. No. K1089, Invitrogen) was prepared using β-lactamase loading solution (Cat. No. K1085, Invitrogen) and 20 μl / well was added to the cells. The substrate solution was incubated in the dark at RT for 2 hours.

[0373] Finally, the fluorescence wavelengths were determined using a Spectra Max (M4) reader (Molecular devices) at the following wavelengths (Ex / Em=409 nm / 460 nm=blue*; Ex / Em=409 nm / 530 nm=green**) and the ratio of blue / green fluorescence after subtraction of the assay medium control was calculated according to the following formula: ratio=(sample-blue*-control-blue*) / (sample-green**-control-green**).

[0374] After pH manipulation, we characterized the final LO candidates (CKLO1-4) for their ability to inhibit CCL2-induced CCR2 signaling, where neutralizing properties were assessed exclusively by monomeric variants of the rec.CCL2 protein used at a final concentration of approximately 15 ng / ml. [Table 18]

[0375] Evaluation of human CCL2 immune complex scavenging by biparatopic antibodies in mice To evaluate the ability of biparatopic antibodies to form immune complexes with wild-type human CCL2, preformed immune complexes consisting of anti-CCL2 biparatopic antibodies (20 mg / kg) and wild-type human CCL2 (0.1 mg / kg) were administered at a single dose of 10 ml / kg into the tail vein of Balb / c mice. Blood was collected 5 min, 7 h, 1 day, 3 days, and 7 days after administration. Serum was prepared by immediately centrifuging the blood at 14,000 rpm for 10 min at 4°C. Serum was stored at -80°C or below until measurement. The biparatopic antibodies tested are listed in Table 4 below. Antibodies with WT IgG1 Fc have Fc gamma receptor binding similar to wild-type, whereas antibodies with PGLALA Fc are Fc gamma receptor binding silent. The results are shown in Figure 4a-i.

[0376] As shown in Figure 4a-i, the immune complex sweeping effect of each anti-CCL2 biparatopic antibody on hCCL2 clearance in vivo was evaluated by comparing anti-CCL2 antibody with Fc gamma receptor binding (solid line) and anti-CCL2 antibody with Fc gamma receptor binding silent (PGLALA, dotted line). Antibody profiles were analyzed by noncompartmental analysis using Phoenix 64 (Pharsight / Certara). AUCinf was estimated by the linear log trapezoidal rule extrapolated to infinity. The clearance value is defined as dose / AUCinf. This clearance difference was also expressed as a fold change calculated by dividing the hCCL2 clearance of the antibody with Fc gamma receptor binding (SG1) by the hCCL2 clearance of the antibody with Fc gamma receptor binding silent (PGLALA) (Table 4 below). The data in Table 4 below show that the clearance of human CCL2 by Fc gamma receptor (FcgR) binding antibodies (WT IgG1) was superior to that by Fc gamma receptor binding silent antibodies (PGLALA (Kabat EU numbering) with IgG1 Fc domain containing mutations L234A, L235A, P329G mutations) for all biparatopic antibodies tested. This suggests that immune complex-mediated scavenging of CCL2 achieved by the biparatopic antibodies tested was more efficient. Furthermore, some biparatopic antibodies showed large fold changes in clearance values, e.g., CNTO / / humanized 11K2 (CNTO / / 11K2).

[0377] Fold change is calculated by dividing the hCCL2 clearance of the antibody with WT Fc gamma R (FcgR) binding by the hCCL2 clearance of the antibody with PGLALA. As shown in Figures 4a-i and Table 4 below, CNTO / / 11k2 shows a maximum fold change of 21.5 between the antibody with IgG1 wild type (WT) with FcgR binding and the antibody that is FcgR binding silent (PGLALA). This suggests that immune complex-mediated scavenging by CNTO / / 11k2-WT IgG1 is the most efficient of all variants. [Table 19]

[0378] Measurement of total human CCL2 concentrations in serum by electrochemiluminescence (ECL) The concentration of total human CCL2 in mouse serum was measured by ECL. 3ug / mL of anti-CCL2 antibody 2F2-SG1 was immobilized overnight on a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and then incubated in blocking buffer at 30°C for 2 hours. Human CCL2 calibration curve samples, quality control samples, and diluted mouse serum samples were incubated with denaturing buffer consisting of 9% SDS at 37°C for 30 minutes and with pH 2.0-2.5 glycine HCl buffer at 37°C for 10 minutes. The purpose of the denaturing buffer is to dissociate human CCL2 from the biparatopic antibody. The samples were then diluted 10-fold and added to the anti-CCL2 immobilized plate and allowed to bind at 30°C for 1 hour, followed by washing. Next, SULFO TAG-labeled MCP-1 antibody was added and the plate was incubated at 30°C for 1 hour, followed by washing. Readout Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal was detected by a SECTOR Imager 2400 (Meso Scale Discovery). Human CCL2 concentrations were calculated based on the standard curve response using the analysis software SOFTmax PRO (Molecular Devices).

[0379] Measurement of serum anti-CCL2 antibody concentrations by enzyme-linked immunosorbent assay (ELISA) The concentration of anti-CCL2 antibody in mouse serum was measured by ELISA. Anti-human IgG κ chain (Antibody Solutions) was dispensed into a Nunc MaxiSorp plate (Thermofisher) and left to stand overnight at 4°C to prepare an anti-human IgG immobilized plate. A calibration curve and samples were prepared with 1% pooled mouse serum. Then, the samples were dispensed into the anti-human IgG immobilized plate and left to stand at 30°C for 1 hour. Then, goat anti-human IgG (gamma chain specific) with HRP conjugate (Southern Biotech) was added and reacted at 30°C for 1 hour. A color reaction was performed using ABTS substrate (KPL) as a substrate, and the absorbance at 450 nm was measured using a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

[0380] Research Overview To summarize the mouse PK study data, the clearance of human CCL2 by the tested biparatopic antibodies was more efficient compared to the monoparatopic antibodies at the same dose. For monoparatopic antibodies, the difference in antigen clearance between WT FcgR-binding and FcgR-silent antibodies was minimal (Table 3). In contrast, a large difference in antigen clearance between WT FcgR-binding and biparatopic antibodies without FcgR binding was obtained (Table 4), suggesting that the tested biparatopic antibodies can efficiently clear human CCL2. The CNTO888 / / 11K2 combination was selected for further antibody engineering as it showed the greatest potential in clearance.

[0381] Example B-2 Anti-CCL2 antibodies with altered variable domains and CDRs (ion-dependent / pH-dependent binding) Modifications resulting in ion- and pH-dependent binding To generate pH-dependent anti-CCL2 antibodies, histidine-scanning mutagenesis was performed on all CDRs of mAb CNTO888 and humanized 11K2. Each amino acid in the CDR was individually mutated to histidine using the In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio company) according to the manufacturer's instructions. After confirming that each variant was correctly mutated by sequencing, the variants were transiently expressed and purified by the following method: Recombinant antibodies were transiently expressed using Freestyle FS293-F cells and 293Fectin (Life technologies) according to the manufacturer's instructions. Recombinant antibodies were purified with Protein A (GE Healthcare) and eluted with D-PBS and His buffer (20 mM histidine, 150 mM NaCl, pH 6.0). For antibodies that are difficult to purify with Protein A, for example, kappaSelect and LambdaFABselect (GE Healthcare) and CaptureSelect IgG-CH1 Affinity Matrix (Thermofisher Scientific) can be used. Size exclusion chromatography was further performed to remove high and / or low molecular weight components as necessary. All histidine-substituted variants were evaluated by a modified BIACORE® assay compared to the one above. Briefly, an additional dissociation phase at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. This is to evaluate the pH-dependent dissociation between antibody (Ab) and antigen (Ag) from the complex formed at pH 7.4 in contrast to the corresponding dissociation at pH 5.8. The dissociation rate in pH 5.8 buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0382] Single histidine substitutions that resulted in a decrease in binding response at pH 5.8 compared to the dissociation phase at pH 7.4 were selected and combined. To identify mutations that improve affinity at pH 7.4, more than 500 variants were generated for each of the heavy and light chains using at least one variant generated during the histidine substitution process. These variants replaced each amino acid in the CDR with the original amino acid and 18 other amino acids except cysteine. The binding ability of the variants to human CCL2 was evaluated at 37°C under pH 7.4 using a BIACORE® 4000 instrument (GE Healthcare). As before, an additional dissociation phase at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. The dissociation rate in pH 5.8 buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0383] Variants with improved affinity at pH 7.4 and pH dependency were selected and these mutations were combined, as exemplified by the four 11K2 variants and the four CNTO888 variants in the table below. [Table 20]

[0384] To evaluate the combinatorial effects of modified 11K2 and CNTO888 variants, each 11K2 variant was combined with four modified CNTO888 variants and expressed as biparatopic CCL2 antibodies in CrossMab format, as illustrated in the table below, where the 4x4 combinations result in the generation of 16 biparatopic antibodies, designated CKLO01-CKLO16. [Table 21]

[0385] To generate bispecific antibodies, the CrossMab technology described in WO 2016 / 016299 was used, in which the VH / VL are exchanged in one antibody arm and the CH1 / CL interface of the other antibody arm is modified by charge modification in combination with knobs-into-holes technology at the CH3 / CH3 interface to promote heterodimerization. Exemplary sequences of all four antibody chains to which this technology is applied are shown for CKLO2IgG1 (see SEQ ID NO: 108 to SEQ ID NO: 111). Depending on the heavy chain constant domain used (e.g., IgG1 wild type (no Fc receptor binding silencing mutations), PGLALA, SG1095, SG1099, 1100 - see description below and sequence description for SG1095, SG1099, 1100), the suffixes IgG1, PGLALA, SG1095, SG1099, 1100 are added.

[0386] Functional characterization of a biparatopic anti-CCL2 antibody with engineered variable domains and CDRs (ion-dependent / pH-dependent binding) Affinity measurements (see methods above) All 16 bispecific anti-CCL2 antibodies generated as IgG1 wild type were determined for their pH-dependent binding to human CCL2.

[0387] FIG. 5a shows Biacore® sensorgrams showing the binding profiles of the four modified 11K2 and four CNTO888 variants after combination to monomeric CCL2 at pH 7.4 (black lines) and pH 5.8 (grey lines) and the 16 Crossmabs.

[0388] Figure 5b shows Biacore® sensorgrams showing the binding profiles of four modified 11K2 and four CNTO888 variants, as well as the 16 Crossmabs after combination with monomeric CCL2, where an additional dissociation phase at pH 5.8 was incorporated into the BIACORE® assay immediately after the dissociation phase at pH 7.4.

[0389] Cross-reactive binding to CCL8 The pH-dependent binding to recombinant monomeric human CCL2 and recombinant monomeric human CCL8 was evaluated at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on each flow cell of a CM4 sensor chip using an amine coupling kit (GE Healthcare) following the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES pH 7.4 or pH 5.8 buffer (20 mM ACES, 150 mM NaCl, 1 mg / ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibodies were captured on the anti-Fc sensor surface, and then recombinant monomeric human CCL2 was injected over the flow cell at 8 nM concentration. The association phase of the analyte to the antibody was monitored for 120 seconds, followed by the dissociation phase for 180 seconds. The sensor surface was regenerated with 3 M MgCl2 for each cycle. Binding sensorgrams were processed by TIBCO Spofire by normalization of binding responses to capture levels.

[0390] The pH-dependent dissociation of the antibody / antigen complex formed at pH 7.4 was evaluated by a modified Biacore assay. Briefly, an additional dissociation phase at pH 5.8 was incorporated into the Biacore assay immediately after the dissociation phase at pH 7.4. The binding sensorgrams were processed by TIBCO Spotfire by normalizing the binding response to the capture level.

[0391] Expression and purification of recombinant human CCL 8 P8A monomer: The sequence of wild-type human CCL8 was obtained from Genbank (NCBI: NP_005614.2). To generate monomeric CCL8, the proline at position 8 of the mature CCL8 protein was mutated to alanine. Expi 293 cells (Lifetech) were transfected according to the manufacturer's instructions. CCL8 wild-type and P8A monomeric proteins were purified using the same method from cell culture supernatants by cation exchange chromatography using SP-Sepharose HP (GE Healthcare) and Superose 200 size exclusion (GE Healthcare) chromatography. Briefly, cell culture supernatants were diluted 2.5-fold with MilliQ water (Millipore), loaded onto a Hi-Trap SP-HP column equilibrated with PBS, washed with equilibration buffer, and eluted using a gradient of 0 to 2 M NaCl. The eluted protein fractions were pooled and further purified by size-exclusion chromatography using a HiLoad 16 / 600 Superose 200 (GE Healthcare) column equilibrated with 20 mM histidine, 150 mM NaCl, pH 6.0. Fractions were analyzed by size-exclusion chromatography and SDS-PAGE. Fractions containing CCL8 protein were pooled, concentrated, and stored at -80°C.

[0392] Human CCL8 shares a high degree of homology with CCL2 and can also bind to CCR2. Because the 11K2 arm can bind to CCL8 (see Figure 1), it was necessary to identify mutations that would reduce this binding to avoid possible off-target effects of neutralizing CCL8. Furthermore, the elimination of CCL8 binding on the 11K2 arm is important for efficient formation of immune complexes with CCL2. Because the CNTO arm does not bind to CCL8, binding of CCL8 to the 11K2 arm may interfere with immune complex formation with CCL2 and reduce the clearance rate of CCL2 from plasma.

[0393] To identify mutations that reduce the binding of 11K2 to human CCL8 and confer selectivity for human CCL2, several CDR positions were substituted to remove cross-reactivity to huCCL8, such as D101E in 11K2 VH of CKLO02 and W92R in 11K2 VL of CKLO03. As shown in Figure 6, CCL8 binding in biparatopic Crossmab could be significantly reduced by engineering 11K2. CKLO01 variant was not optimized to reduce CCL8 binding, but CKLO04, CKLO03, and CKLO02 contain mutations that reduce CCL8 binding. All four Crossmab have pH-dependent binding to CCL8.

[0394] Binding affinity of anti-CCL2 antibodies to recombinant CCL2 and CCL8 at pH 7.4 and pH 5.8 To determine the affinity and pH-dependent binding of parent CNTO888H / 11K2H2, CKLO1, CKLO2 and CKLO3 to human CCL2 and CCL8, they were evaluated at 37 °C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on each flow cell of a CM4 sensor chip using an amine coupling kit (GE Healthcare) following the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES pH 7.4 or pH 5.8 buffer (20 mM ACES, 150 mM NaCl, 1 mg / ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibodies were captured on the anti-Fc sensor surface and then recombinant human CCL2 P8A variant (monomer) and CCL8 P8A variant (monomer) were injected over the flow cells at 1.25 nM to 20 nM prepared by two-fold serial dilutions. The sensor surface was regenerated with 3M MgCl2 after each cycle. Data was processed using Biacore T200 Evaluation software, version 2.0 (GE Healthcare), and binding affinities were determined by fitting to a 1:1 binding model. The binding affinities of anti-CCL2 antibodies to recombinant CCL2 and CCL8 at pH 7.4 and pH 5.8 are shown in Table 5 below. [Table 22]

[0395] The data in Table 5 show that binding to CCL8 at pH 7.4 was abolished for CKLO2 and CKLO3, while CCL2 maintained strong affinity and pH-dependent binding at pH 7.4. The results show that different modifications introduced into the variable regions and CDRs of the parent bispecific antibodies based on CNTO888 and 11K2 successfully generated affinity matured variants, CKLO1, CKLO2, CKLO3, with enhanced binding affinity to CCL2 at pH 7.4 compared to the parent Ab. At the same time, CKLO1, CKLO2, CKLO3 showed strong pH-dependent binding to CCL2. Compared to the KD value at pH 5.8, a weaker KD of more than 1000-fold lower binding affinity to CCL2 was observed at pH 7.4.

[0396] Clearance of wild-type human CCL2 To evaluate the ability of pH-dependent bispecific antibodies to enhance clearance of wild-type human CCL2, preformed immune complexes consisting of anti-CCL2 monoparatopic antibodies (20 mg / kg) and wild-type human CCL2 (0.1 mg / kg) were administered at a single dose of 10 ml / kg into the tail vein of SCID mice. Blood was collected 5 min, 1 h, 4 h, 7 h, 1 day, and 7 days after administration. Serum was stored at or below -80°C until assayed. Crossmab antibodies tested were the parent CNTO / / 11K2, and four pH-engineered variants, CKLO01, CKLO02, CKLO03, and CKLO04. All antibodies had an IgG1 wild-type Fc portion (no mutations to silence / abolish Fc (gamma) receptor binding). Measurement of total human CCL2 and anti-CCL2 antibody concentrations in mouse serum was performed as described above (Table 4, under "Evaluation of human CCL2 immune complex scavenging in mice using biparatopic antibodies").

[0397] Results are shown in Figure 7a: Serum concentration of hCCL2 over time after injection of preformed immune complexes consisting of hCCL2 and bispecific anti-CCL2 antibodies (parental CNTO / / 11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03 and CKLO04) into SCID mice. All four pH-engineered variants showed rapid clearance of human CCL2. For CKLO02, CKLO03, human CCL2 was below the limit of detection by day 1. For parental CNTO / / 11K2, rapid clearance of human CCL2 was initially observed by day 1, but thereafter the clearance of human CCL 2 was slo...

Claims

1. A bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second, different epitope on human CCL2, wherein the bispecific antibody a) a first polypeptide chain comprising (in the N-terminal to C-terminal direction) VH1-CH1-L1-hinge-CH2-CH3-L2-VL1-CL, wherein VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both together forming the first antigen-binding site), CH1 is a constant heavy chain domain 1, L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one claim, 5 to 10 amino acids), hinge is a heavy chain hinge region, CH2 is a constant heavy chain domain 2, CH3 is a constant heavy chain domain 3, L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one claim, 10 to 15 amino acids), CL is a constant light chain domain, the first polypeptide chain, and b) a second polypeptide chain comprising (in the N-terminal to C-terminal direction) VH2-CH1-L1-hinge-CH2-CH3-L2-VL2-CL, wherein VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both together forming the second antigen-binding site), CH1 is a constant heavy chain domain 1, L1 is a polypeptide linker having a length of 5 to 15 amino acids (in one claim, 5 to 10 amino acids), hinge is a heavy chain hinge region, CH2 is a constant heavy chain domain 2, CH3 is a constant heavy chain domain 3, L2 is a polypeptide linker having a length of 5 to 15 amino acids (in one claim, 10 to 15 amino acids), CL is a constant light chain domain, the second polypeptide chain, and comprises A) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or B) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 91, the VL2 domain contains the amino acid sequence of SEQ ID NO: 93, or or C) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 71, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 90, the VL2 domain contains the amino acid sequence of SEQ ID NO: 94, or or D) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 72, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 90, the VL2 domain contains the amino acid sequence of SEQ ID NO: 94, or or E) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 73, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 90, the VL2 domain contains the amino acid sequence of SEQ ID NO: 93, or or F) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 73, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 90, the VL2 domain contains the amino acid sequence of SEQ ID NO: 94, or or G) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 73, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 92, the VL2 domain contains the amino acid sequence of SEQ ID NO: 93, or or H) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 73, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 91, the VL2 domain contains the amino acid sequence of SEQ ID NO: 93, or or I) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 72, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain contains the amino acid sequence of SEQ ID NO: 90, the VL2 domain contains the amino acid sequence of SEQ ID NO: 93, or or J) i) the VH1 domain contains the amino acid sequence of SEQ ID NO: 72, the VL1 domain contains the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or K) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or L) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or M) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94, or or N) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or O) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, or or P) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, bispecific antibody. **Claim 2** i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71, the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 91, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 93, the bispecific antibody according to claim 1. **Claim 3** i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 71, The VL1 domain comprises the amino acid sequence of SEQ ID NO: 75, ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 90, the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94, The bispecific antibody according to claim 1.

4. L1 is a polypeptide linker having a length of 9 to 11 amino acids, L2 is a polypeptide linker having a length of 9 to 11 amino acids, The bispecific antibody according to any one of claims 1 to 3.

5. L1 and L2 are GSGGGSGGGG (SEQ ID NO: 183), GSGGGGGSGG (SEQ ID NO: 184), GSGGGGGGSGG (SEQ ID NO: 185), GGSGSGGGGG (SEQ ID NO: 186), GGSGGGGGSG (SEQ ID NO: 187), GGSGGGGGSG (SEQ ID NO: 188), GGGSGSGGGG (SEQ ID NO: 189), GGGSGGGGGS (SEQ ID NO: 190), and GGGGSGSGGG (SEQ ID NO: 191) a polypeptide linker selected from the group of The bispecific antibody according to claim 4.

6. L1 is a polypeptide linker comprising the amino acid sequence of GGSGGGGGSG (SEQ ID NO: 188), L2 is a polypeptide linker comprising the amino acid sequence of GGSGGGGGSG (SEQ ID NO: 188), The bispecific antibody according to claim 4.

7. The constant heavy chain domains CH1, hinge, CH2 and CH3 are of human IgG isotype, preferably human IgG1 isotype, the bispecific antibody according to any one of claims 1 to 3.

8. The bispecific antibody i) Block the binding of CCL2 to its receptor CCR2 in vitro (reporter assay, IC ５０ = 0.5 nM), and / or ii) inhibiting the CCL2-mediated chemotaxis of myeloid cells in vitro (IC ５０ = 1.5 nM), and / or iii) is cross-reactive with cynomolgus (cyno) and human CCL2, The bispecific antibody according to any one of claims 1 to 3.

9. The bispecific antibody is not cross-reactive with other CCL homologs selected from the group of CCL8, CCL7 and CCL13 as compared to the binding to CCL2, the bispecific antibody according to any one of claims 1 to 3.

10. The bispecific antibody binds to human CCL2 in a pH-dependent manner, and both the first antigen-binding site and the second antigen-binding site bind to CCL2 with higher affinity at neutral pH than at acidic pH, the bispecific antibody according to any one of claims 1 to 3.

11. The bispecific antibody according to any one of claims 1 to 3, wherein the bispecific antibody binds to human CCL2 with an affinity 10-fold higher than that at pH 5.8 at pH 7.

4.

12. The constant heavy chain domains CH1, hinge, CH2, and CH3 are of the human IgG1 isotype and have the following mutations (Kabat EU numbering): i) Q311R and / or P343R, and / or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T, and / or A330K, and / or iii) M428L, N434A, and / or Y436T, and / or iv) Q438R and / or S440E The bispecific antibody according to any one of claims 1 to 3, comprising one or more of the above.

13. The constant heavy chain domains CH1, hinge, CH2, and CH3 are of the human IgG1 isotype and have the following mutations (Kabat EU numbering): i) Q311R and / or P343R, and / or ii) L235W, G236N, H268D, Q295L, K326T, and / or A330K, and / or iii) N434A, and / or iv) Q438R and / or S440E The bispecific antibody according to any one of claims 1 to 3, comprising one or more of the above.

14. The constant heavy chain domains CH1, hinge, CH2, and CH3 are of the human IgG1 isotype and have the following mutations (Kabat EU numbering): i) Q311R and P343R, and ii) L234Y, P238D, T250V, V264I, T307V, and A330K, and iii) M428L, N434A, and Y436T, and iv) Q438R and S440E The bispecific antibody according to any one of claims 1 to 3, comprising one or more of the above.

15. The constant heavy chain domain CH3 has the following mutation (Kabat EU numbering) K447G The bispecific antibody according to claim 12, comprising the above.

16. A nucleic acid encoding the antibody according to any one of claims 1 to 3.

17. A host cell comprising the nucleic acid according to claim 16.

18. A method for producing an antibody, comprising culturing the host cell according to claim 17 so that the antibody is produced.

19. The method according to claim 18, further comprising recovering the antibody from the host cell.

20. A pharmaceutical preparation comprising a bispecific antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

21. A bispecific antibody according to any one of claims 1 to 3 for use as a medicament.

22. A bispecific antibody according to any one of claims 1 to 3 for use in the treatment of cancer, or an inflammatory or autoimmune disease.

23. Use of a bispecific antibody according to any one of claims 1 to 3 in the manufacture of a medicament.

24. The use according to claim 23, wherein the medicament is for the treatment of cancer.

25. The use according to claim 24, wherein the medicament is for the treatment of an inflammatory or autoimmune disease.

26. A method of treating an individual having cancer, the method comprising administering to the individual an effective amount of a bispecific antibody according to any one of claims 1 to 3.

27. A method of treating an individual having an inflammatory or autoimmune disease, the method comprising administering to the individual an effective amount of a bispecific antibody according to any one of claims 1 to 3.